Novel human membrane proteins and polynucleotides encoding the same

ABSTRACT

The nucleotide and amino acid sequences of several novel human G protein coupled receptors are described.

[0001] The present application claims the benefit of U.S. ProvisionalApplication Number 60/169,427 which was filed on Dec, 7, 1999 and isherein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identificationand characterization of novel human polynucleotides that encode membraneassociated proteins and receptors. The invention encompasses thedescribed polynucleotides, host cell expression systems, the encodedproteins, fusion proteins, polypeptides and peptides, antibodies to theencoded proteins and peptides, and genetically engineered animals thatlack the disclosed sequences, or over express the disclosed sequences,or antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed sequences that can be used for diagnosis, drug screening,clinical trial monitoring, and/or the treatment of physiological orbehavioral disorders.

2. BACKGROUND OF THE INVENTION

[0003] Membrane receptor proteins are integral components of themechanisms through which cells sense their surroundings as well asmaintain cellular homeostasis and function. Accordingly, membranereceptor proteins are often involved in signal transduction pathwaysthat control cell physiology, chemical communication, and geneexpression. A particularly relevant class of membrane receptors arethose typically characterized by the presence of 7 conservedtransmembrane domains that are interconnected by nonconservedhydrophilic loops. Such, “7TM receptors” include a superfamily ofreceptors known as G-protein coupled receptors (GPCRs). GPCRs aretypically involved in signal transduction pathways involving G-proteinsor PPG proteins. As such, the GPCR family includes many receptors thatare known to serve as drug targets for therapeutic agents.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel GPCRs, and thecorresponding novel GPCR (NGPCR) amino acid sequences. The NGPCRsdescribed for the first time herein, are transmembrane proteins thatspan the cellular membrane and are involved in signal transduction afterligand binding. The described NGPCRs have structural motifs found in the7TM receptor family. Expression of the described NGPCRs can be detectedin human spleen, bone marrow, and adipose, cells, among others. Thenovel human GPCRs described herein encode proteins of 225, 508, 298,359, 233, 162, 504, 294, 355, 229, 158, 521, 311, 372, 246, 175, 485,275, 336, 210, 139, 549, 339, 400, 274, 203, amino acids in length (seerespectively SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52). The describedNGPCRs have a characteristic leader sequence, and contain thecharacteristic multiple transmembrane regions (of about 20-30 aminoacids), as well as several predicted cytoplasmic domains.

[0005] Additionally contemplated are “knockout” ES cells that have beenproduced using conventional methods (see, for example, PCT Applic. No.PCT/US98/03243, filed Feb. 20, 1998, herein incorporated by reference).Accordingly, an additional aspect of the present invention includesknockout cells and animals having genetically engineered mutations inthe sequence encoding the presently described NGPCRs.

[0006] The invention encompasses the nucleotides presented in theSequence Listing, host cells expressing such nucleotides, and theexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described NGPCRs, including thespecifically described human NGPCRS, and the human NGPCR sequenceproducts; (b) nucleotides that encode one or more portions of the NGPCRsthat correspond to functional domains, and the polypeptide productsspecified by such nucleotide sequences, including but not limited to thenovel regions of the described extracellular domain(s) (ECD), one ormore transmembrane domain(s) (TM) first disclosed herein, and thecytoplasmic domain(s) (CD); (c) isolated nucleotides that encodemutants, engineered or naturally occurring, of the described NGPCRs inwhich all or a part of at least one of the domains is deleted oraltered, and the polypeptide products specified by such nucleotidesequences, including but not limited to soluble receptors in which allor a portion of the TM is deleted, and nonfunctional receptors in whichall or a portion of the CD is deleted; (d) nucleotides that encodefusion proteins containing the coding region from an NGPCR, or one ofits domains (e.g., an extracellular domain) fused to another peptide orpolypeptide.

[0007] The invention also encompasses agonists and antagonists of theNGPCRs, including small molecules, large molecules, mutant NGPCRproteins, or portions thereof that compete with the native NGPCR, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NGPCR (e.g., antisense and ribozymemolecules, and gene or regulatory sequence replacement constructs) or toenhance the expression of the described NGPCR sequence (e.g., expressionconstructs that place the described sequence under the control of astrong promoter system), and transgenic animals that express a NGPCRtransgene or “knock-outs” that do not express a functional NGPCR.

[0008] Further, the present invention also relates to methods for theuse of the described NGPCR sequence and/or NGPCR gene products for theidentification of compounds that modulate, i.e., act as agonists orantagonists, of NGPCR gene expression and or NGPCR gene productactivity. Such compounds can be used as therapeutic agents for thetreatment of various symptomatic representations of biological disordersor imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequence of 26 NGPCR ORFs, theamino acid sequences encoded thereby, as well as an ORF with surrounding5′ and 3′ regions (SEQ ID NO:53).

5. DETAILED DESCRIPTION OF THE INVENTION

[0010] The human NGPCRS, described for the first time herein, are novelreceptor proteins that are expressed in human cells. The described NGPCRsequences were obtained using sequences from gene trapped human cellsand cDNA clones isolated from human lymph node and bone marrow cDNAlibraries (Edge Biosystems, Gaithersburg, Md.). The described NGPCRs aretransmembrane proteins that fall within the 7TM family of receptors. Aswith other GPCRs, signal transduction is triggered when a ligand bindsto the receptor. Interfering with the binding of the natural ligand, orneutralizing or removing the ligand, or interference with its binding toa NGPCR will effect NGPCR mediated signal transduction. Because of theirbiological significance, 7TM, and particularly GPCR, proteins have beensubjected to intense scientific/commercial scrutiny (see, for example,U.S. Application Ser. Nos. 08/820,521, filed Mar. 19, 1997, and08/833,226, filed Apr. 17, 1997 both of which are herein incorporated byreference in their entirety for applications, uses, and assays involvingthe described NGPCRs).

[0011] The invention encompasses the use of the described NGPCRnucleotides, NGPCR proteins and peptides, as well as antibodies,preferably humanized monoclonal antibodies, or binding fragments,domains, or fusion proteins thereof, to the NGPCRs (which can, forexample, act as NGPCR agonists or antagonists), antagonists that inhibitreceptor activity or expression, or agonists that activate receptoractivity or increase its expression in the diagnosis and treatment ofdisease.

[0012] In particular, the invention described in the subsections belowencompasses NGPCR polypeptides or peptides corresponding to functionaldomains of NGPCR (e.g., ECD, TM or CD), mutated, truncated or deletedNGPCRs (e.g., NGPCRs missing one or more functional domains or portionsthereof, such as, AECD, ATM and/or ACD), NGPCR fusion proteins (e.g., aNGPCR or a functional domain of a NGPCR, such as the ECD, fused to anunrelated protein or peptide such as an immunoglobulin constant region,i.e., IgFc), nucleotide sequences encoding such products, and host cellexpression systems that can produce such NGPCR products.

[0013] The invention also encompasses antibodies and anti-idiotypicantibodies (including Fab fragments), antagonists and agonists of theNGPCR, as well as compounds or nucleotide constructs that inhibitexpression of a NGPCR gene (transcription factor inhibitors, antisenseand ribozyme molecules, or gene or regulatory sequence replacementconstructs), or promote expression of NGPCR (e.g., expression constructsin which NGPCR coding sequences are operatively associated withexpression control elements such as promoters, promoter/enhancers,etc.). The invention also relates to host cells and animals geneticallyengineered to express the human NGPCRs (or mutants thereof) or toinhibit or “knock-out” expression of the animal's endogenous NGPCRgenes.

[0014] The NGPCR proteins or peptides, NGPCR fusion proteins, NGPCRnucleotide sequences, antibodies, antagonists and agonists can be usefulfor the detection of mutant NGPCRs or inappropriately expressed NGPCRsfor the diagnosis of disease. The NGPCR proteins or peptides, MGPCRfusion proteins, NGPCR nucleotide sequences, host cell expressionsystems, antibodies, antagonists, agonists and genetically engineeredcells and animals can be used for screening for drugs (or highthroughput screening of combinatorial libraries) effective in thetreatment of the symptomatic or phenotypic manifestations of perturbingthe normal function of NGPCR in the body. The use of engineered hostcells and/or animals may offer an advantage in that such systems allownot only for the identification of compounds that bind to an ECD of aNGPCR, but can also identify compounds that affect the signal transducedby an activated NGPCR.

[0015] Finally, the NGPCR protein products (especially solublederivatives such as peptides corresponding to the NGPCR ECD, ortruncated polypeptides lacking on or more TM domains) and fusion proteinproducts (especially NGPCR-Ig fusion proteins, i.e., fusions of a NGPCR,or a domain of a NGPCR, e.g., ECD, ΔTM to an IgFc), antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists oragonists (including compounds that modulate signal transduction whichmay act on downstream targets in a NGPCR-mediated signal transductionpathway) can be used for therapy of such diseases. For example, theadministration of an effective amount of soluble NGPCR ECD, ΔTM, or anECD-IgFc fusion protein or an anti-idiotypic antibody (or its Fab) thatmimics the NGPCR ECD would “mop up” or “neutralize” the endogenous NGPCRligand, and prevent or reduce binding and receptor activation.Nucleotide constructs encoding such NGPCR products can be used togenetically engineer host cells to express such products in vivo; thesegenetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NGPCR, a NGPCR peptide, soluble ECDor ATM or a NGPCR fusion protein that will “mop up” or neutralize aNGPCR ligand. Nucleotide constructs encoding functional NGPCRs, mutantNGPCRs, as well as antisense and ribozyme molecules can be used in “genetherapy” approaches for the modulation of NGPCR expression. Thus, theinvention also encompasses pharmaceutical formulations and methods fortreating biological disorders.

[0016] Various aspects of the invention are described in greater detailin the subsections below.

5.1 The NGPCR Genes

[0017] The cDNA sequences and deduced amino acid sequences of thedescribed human NGPCRs are presented in the Sequence Listing.

[0018] The NGPCRs of the present invention include: (a) the human DNAsequences presented in the Sequence Listing and additionally contemplateany nucleotide sequence encoding a contiguous and functional NGPCR openreading frame (ORF) that hybridizes to a complement of the DNA sequencespresented in the Sequence Listing under highly stringent conditions,e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1× SSC/0.1%SDS at 68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & sons, Inc., New York, at p. 2.10.3) and encodes a functionallyequivalent gene product. Additionally contemplated are any nucleotidesequences that hybridize to the complement of the DNA sequences thatencode and express an amino acid sequence presented in the SequenceListing under moderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet which stillencode a functionally equivalent NGPCR gene product. Functionalequivalents of NGPCR include naturally occurring NGPCRs present in otherspecies, and mutant NGPCRs whether naturally occurring or engineered.The invention also includes degenerate variants of the disclosedsequences.

[0019] Additionally contemplated are polynucleotides encoding NGPCRORFs, or their functional equivalents, encoded by polynucleotidesequences that are about 99, 95, 90, or about 85 percent similar oridentical to corresponding regions of the polynucleotide sequencesdescribed in the Sequence Listing (as measured by BLAST sequencecomparison analysis using, for example, the GCG sequence analysispackage using default parameters).

[0020] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NGPCR nucleotide sequences. Such hybridization conditionsmay be highly stringent or less highly stringent, as described above. Ininstances wherein the nucleic acid molecules are deoxyoligonucleotides(“DNA oligos”), such molecules (and particularly about 16 to about 100base long, about 20 to about 80, or about 34 to about 45 base long, orany variation or combination of sizes represented therein incorporatinga contiguous region of sequence first disclosed in the present SequenceListing, can be used in conjunction with the polymerase chain reaction(PCR) to screen libraries, isolate clones, and prepare cloning andsequencing templates, etc. Alternatively, the oligonucleotides can beused singly or in chip format as hybridization probes. For example, aseries of the described NGPCR oligonucleotide sequences, or thecomplements thereof, can be used to represent all or a portion of thedescribed NGPCRs. The oligonucleotides, typically between about 16 toabout 40 (or any whole number within the stated range) nucleotides inlength may partially overlap each other and/or the NGPCR sequence may berepresented using oligonucleotides that do not overlap. Accordingly, thedescribed NGPCR polynucleotide sequences shall typically comprise atleast about two or three distinct oligonucleotide sequences of at leastabout 18 nucleotides in length that are each first disclosed in thedescribed Sequence Listing. Such oligonucleotide sequences may begin atany nucleotide present within a sequence in the Sequence Listing andproceed in either a sense (5′-to-3′) orientation vis-a-vis the describedsequence or in an antisense orientation. For oligonucleotides probes,highly stringent conditions may refer, e.g., to washing in 6× SSC/0.05%sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).

[0021] The described oligonucleotides may encode or act as NGPCRantisense molecules, useful, for example, in NGPCR gene regulation (forand/or as antisense primers in amplification reactions of NGPCR nucleicacid sequences). With respect to NGPCR gene regulation, such techniquescan be used to regulate biological functions. Further, such sequencesmay be used as part of ribozyme and/or triple helix sequences, alsouseful for NGPCR gene regulation.

[0022] Additionally, the antisense oligonucleotides may comprise atleast one modified base moiety which is selected from the groupincluding but not limited to 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0023] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0024] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0025] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0026] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g. by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0027] Low stringency conditions are well known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, N.Y.

[0028] Alternatively, suitably labeled NGPCR nucleotide probes may beused to screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms, determining thegenomic structure of a given locus/allele, and designing diagnostictests. For example, sequences derived from regions adjacent to theintron/exon boundaries of the human gene can be used to design primersfor use in amplification assays to detect mutations within the exons,introns, splice sites (e.g., splice acceptor and/or donor sites), etc.,that can be used in diagnostics and pharmacogenomics.

[0029] Further, a NGPCR sequence homolog may be isolated from nucleicacid of the organism of interest by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequences within the NGPCR product disclosed herein. The template forthe reaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue known or suspected to express a NGPCR gene allele.

[0030] The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NGPCR gene.The PCR fragment may then be used to isolate a full length cDNA clone bya variety of methods. For example, the amplified fragment may be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

[0031] PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express a NGPCR gene). A reverse transcription(RT) reaction may be performed on the RNA using an oligonucleotideprimer specific for the most 5′ end of the amplified fragment for thepriming of first strand synthesis. The resulting RNA/DNA hybrid may thenbe “tailed” using a standard terminal transferase reaction, the hybridmay be digested with RNase H, and second strand synthesis may then beprimed with a complementary primer. Thus, cDNA sequences upstream of theamplified fragment may easily be isolated. For a review of cloningstrategies which may be used, see e.g., Sambrook et al., 1989, supra.

[0032] A cDNA of a mutant NGPCR gene can be isolated, for example, byusing PCR. In this case, the first cDNA strand may be synthesized byhybridizing an oligo-dT oligonucleotide to mRNA isolated from tissueknown or suspected to be expressed in an individual putatively carryinga mutant NGPCR allele, and by extending the new strand with reversetranscriptase. The second strand of the cDNA is then synthesized usingan oligonucleotide that hybridizes specifically to the 5′ end of thenormal gene. Using these two primers, the product is then amplified viaPCR, optionally cloned into a suitable vector, and subjected to DNAsequence analysis through methods well known to those of skill in theart. By comparing the DNA sequence of the mutant NGPCR allele to that ofthe normal NGPCR allele, the mutation(s) responsible for the loss oralteration of function of the mutant NGPCR gene product can beascertained.

[0033] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of or known to carry the mutantNGPCR allele, or a cDNA library can be constructed using RNA from atissue known, or suspected, to express the mutant NGPCR allele. A normalNGPCR gene, or any suitable fragment thereof, can then be labeled andused as a probe to identify the corresponding mutant NGPCR allele insuch libraries. Clones containing the mutant NGPCR gene sequences canthen be purified and subjected to sequence analysis according to methodswell known to those of skill in the art.

[0034] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NGPCR allele in an individual suspectedof or known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against the normal NGPCR gene product, as described, below, inSection 5.3. (For screening techniques, see, for example, Harlow, E. andLane, eds., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborPress, Cold Spring Harbor) Additionally, screening can be accomplishedby screening with labeled NGPCR fusion proteins, such as, for example,alkaline phosphatase-NGPCR or NGPCR-alkaline phosphatase fusionproteins. In cases where a NGPCR mutation results in an expressed geneproduct with altered function (e.g., as a result of a missense or aframeshift mutation), a polyclonal set of antibodies to NGPCR are likelyto cross-react with the mutant NGPCR gene product. Library clonesdetected via their reaction with such labeled antibodies can be purifiedand subjected to sequence analysis according to methods well known tothose of skill in the art.

[0035] The invention also encompasses nucleotide sequences that encodemutant NGPCRs, peptide fragments of the NGPCRs, truncated NGPCRs, andNGPCR fusion proteins. These include, but are not limited to, nucleotidesequences encoding mutant NGPCRs described below; polypeptides orpeptides corresponding to one or more ECD, TM and/or CD domains of theNGPCR or portions of these domains; truncated NGPCRs in which one or twoof the domains is deleted, e.g., a soluble NGPCR lacking the TM or boththe TM and CD regions, or a truncated, nonfunctional NGPCR lacking allor a portion of the CD region. Nucleotides encoding fusion proteins mayinclude, but are not limited to, full length NGPCR sequences, truncatedNGPCRs, or nucleotides encoding peptide fragments of NGPCR fused to anunrelated protein or peptide, such as for example, a transmembranesequence, which anchors the NGPCR ECD to the cell membrane; an IgFcdomain which increases the stability and half life of the resultingfusion protein (e.g., NGPCR-Ig) in the bloodstream; or an enzyme,fluorescent protein, luminescent protein which can be used as a marker.

[0036] The invention also encompasses (a) DNA vectors that contain anyof the foregoing NGPCR coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNGPCR coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing NGPCR codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences in the host cell. As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the humancytomegalovirus (hCMV) immediate early gene, regulatable, viral elements(particularly retroviral LTR promoters), the early or late promoters ofSV40 adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

5.2 NGPCR Proteins and Polypeptides

[0037] NGPCRs, polypeptides, peptide fragments, mutated, truncated, ordeleted forms of the NGPCRS, and/or NGPCR fusion proteins can beprepared for a variety of uses. These uses include, but are not limitedto, the generation of antibodies, as reagents in diagnostic assays, forthe identification of other cellular gene products related to a NGPCR,as reagents in assays for screening for compounds that can be aspharmaceutical reagents useful in the therapeutic treatment of mental,biological, or medical disorders and disease.

[0038] The Sequence Listing discloses the amino acid sequences encodedby the described NGPCR genes. The NGPCRs have initiator methionines inDNA sequence contexts consistent with translation initiation sites,followed by hydrophobic signal sequences typical of membrane associatedproteins. The sequence data presented herein indicate that alternativelyspliced forms of the NGPCRs exist (which may or may not be tissuespecific).

[0039] The NGPCR amino acid sequences of the invention include thenucleotide and amino acid sequences presented in the Sequence Listing aswell as analogues and derivatives thereof. Further, corresponding NGPCRhomologues from other species are encompassed by the invention. In fact,any NGPCR protein encoded by the NGPCR nucleotide sequences describedabove are within the scope of the invention, as are any novelpolynucleotide sequences encoding all or any novel portion of an aminoacid sequence presented in the Sequence Listing. The degenerate natureof the genetic code is well known, and, accordingly, each amino acidpresented in the Sequence Listing, is generically representative of thewell known nucleic acid “triplet” codon, or in many cases codons, thatcan encode the amino acid. As such, as contemplated herein, the aminoacid sequences presented in the Sequence Listing, when taken togetherwith the genetic code (see, for example, Table 4-1 at page 109 of“Molecular Cell Biology”, 1986, J. Darnell et al. eds., ScientificAmerican Books, New York, N.Y., herein incorporated by reference) aregenerically representative of all the various permutations andcombinations of nucleic acid sequences that can encode such amino acidsequences.

[0040] The invention also encompasses proteins that are functionallyequivalent to the NGPCR encoded by the described nucleotide sequences asjudged by any of a number of criteria, including but not limited to theability to bind a ligand for a NGPCR, the ability to effect an identicalor complementary signal transduction pathway, a change in cellularmetabolism (e.g., ion flux, tyrosine phosphorylation, etc.) or change inphenotype when the NGPCR equivalent is present in an appropriate celltype (such as the amelioration, prevention or delay of a biochemical,biophysical, or overt phenotype. Such functionally equivalent NGPCRproteins include but are not limited to additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NGPCRnucleotide sequences described above but which result in a silentchange, thus producing a functionally equivalent gene product. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0041] While random mutations can be made to NGPCR DNA (using randommutagenesis techniques well known to those skilled in the art) and theresulting mutant NGPCRs tested for activity, site-directed mutations ofthe NGPCR coding sequence can be engineered (using site-directedmutagenesis techniques well known to those skilled in the art) togenerate mutant NGPCRs with increased function, e.g., higher bindingaffinity for the target ligand, and/or greater signaling capacity; ordecreased function, and/or decreased signal transduction capacity. Onestarting point for such analysis is by aligning the disclosed humansequences with corresponding gene/protein sequences from, for example,other mammals in order to identify amino acid sequence motifs that areconserved between different species. Non-conservative changes can beengineered at variable positions to alter function, signal transductioncapability, or both. Alternatively, where alteration of function isdesired, deletion or non-conservative alterations of the conservedregions (i.e., identical amino acids) can be engineered. For example,deletion or non-conservative alterations (substitutions or insertions)of the various conserved transmembrane domains.

[0042] An additional application of the described NGPCR polynucleotidesequences is their use in the molecular mutagenesis/evolution ofproteins that are at least partially encoded by the described novelsequences using, for example, polynucleotide shuffling or relatedmethodologies. Such approaches are described in U.S. Pat. Nos. 5,830,721and 5,837,458 which are herein incorporated by reference in theirentirety.

[0043] Other mutations to the NGPCR coding sequence can be made togenerate NGPCRs that are better suited for expression, scale up, etc. inthe host cells chosen. For example, cysteine residues can be deleted orsubstituted with another amino acid in order to eliminate disulfidebridges; N-linked glycosylation sites can be altered or eliminated toachieve, for example, expression of a homogeneous product that is moreeasily recovered and purified from yeast hosts which are known tohyperglycosylate N-linked sites. To this end, a variety of amino acidsubstitutions at one or both of the first or third amino acid positionsof any one or more of the glycosylation recognition sequences whichoccur in the ECD (N-X-S or N-X-T), and/or an amino acid deletion at thesecond position of any one or more such recognition sequences in the ECDwill prevent glycosylation of the NGPCR at the modified tripeptidesequence. (See, e.g., Miyajima et al., 1986, EMBO J. 5(6):1193-1197).

[0044] Peptides corresponding to one or more domains of the NGPCR (e.g.,ECD, TM, CD, etc.), truncated or deleted NGPCRs (e.g., NGPCR in which aECD, TM and/or CD is deleted) as well as fusion proteins in which a fulllength NGPCR, a NGPCR peptide, or truncated NGPCR is fused to anunrelated protein, are also within the scope of the invention and can bedesigned on the basis of the presently disclosed NGPCR nucleotide andNGPCR amino acid sequences. Such fusion proteins include but are notlimited to IgFc fusions which stabilize the NGPCR protein or peptide andprolong half-life in vivo; or fusions to any amino acid sequence thatallows the fusion protein to be anchored to the cell membrane, allowingan ECD to be exhibited on the cell surface; or fusions to an enzyme,fluorescent protein, or luminescent protein which provide a markerfunction.

[0045] Also encompassed by the present invention are novel proteinconstructs engineered in such a way that they facilitate transport ofthe NGPCR to the target site, to the desired organ, across or into thecell membrane and/or to the nucleus where the NGPCR can exert itsfunction activity. This goal may be achieved by coupling of the NGPCR toa cytokine or other ligand that would direct the NGPCR to the targetorgan and facilitate receptor mediated transport across the membraneinto the cytosol. Conjugation of NGPCRs to antibody molecules or theirFab fragments could be used to target cells bearing a particularepitope. Attaching the appropriate signal sequence to the NGPCR wouldalso transport the NGPCR to the desired location within the cell.Alternatively targeting of NGPCR or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in Liposomes:A Practical Approach, New RRCed., Oxford University Press, New York and in U.S. Pat. Nos. 4,594,595,5,459,127, 5,948,767 and 6,110,490 and their respective disclosureswhich are herein incorporated by reference in their entirety.

[0046] While the NGPCR polypeptides and peptides can be chemicallysynthesized (e.g., see Creighton, 1983, Proteins: Structures andMolecular Principles, W.H. Freeman & Co., N.Y.), large polypeptidesderived from a NGPCR and full length NGPCRs can be advantageouslyproduced by recombinant DNA technology using techniques well known inthe art for expressing nucleic acid containing NGPCR gene sequencesand/or coding sequences. Such methods can be used to constructexpression vectors containing a presently described NGPCR nucleotidesequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.See, for example, the techniques described in Sambrook et al., 1989,supra, and Ausubel et al., 1989, supra. Alternatively, RNA correspondingto all or a portion of a transcript encoded by a NGPCR nucleotidesequence may be chemically synthesized using, for example, synthesizers.See, for example, the techniques described in “OligonucleotideSynthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford, which isincorporated by reference herein in its entirety.

[0047] A variety of host-expression vector systems may be utilized toexpress the NGPCR nucleotide sequences of the invention. Where the NGPCRpeptide or polypeptide is a soluble derivative (e.g., NGPCR peptidescorresponding to an ECD; truncated or deleted NGPCR in which a TM and/orCD are deleted) the peptide or polypeptide can be recovered from theculture, i.e., from the host cell in cases where the NGPCR peptide orpolypeptide is not secreted, and from the culture media in cases wherethe NGPCR peptide or polypeptide is secreted by the cells. However, suchexpression systems also encompass engineered host cells that express aNGPCR, or functional equivalent, in situ, i.e., anchored in the cellmembrane. Purification or enrichment of NGPCR from such expressionsystems can be accomplished using appropriate detergents and lipidmicelles and methods well known to those skilled in the art. However,such engineered host cells themselves may be used in situations where itis important not only to retain the structural and functionalcharacteristics of the NGPCR, but to assess biological activity, e.g.,in drug screening assays.

[0048] The expression systems that may be used for purposes of theinvention include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing NGPCRnucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformedwith recombinant yeast expression vectors containing NGPCR nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NGPCR sequences; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing NGPCR nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0049] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NGPCRgene product being expressed. For example, when a large quantity of sucha protein is to be produced, for the generation of pharmaceuticalcompositions of NGPCR protein or for raising antibodies to a NGPCRprotein, for example, vectors that direct the expression of high levelsof fusion protein products that are readily purified may be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which a NGPCRcoding sequence may be ligated individually into the vector in framewith the lacZ coding region so that a fusion protein is produced; PINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.PGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

[0050] In an insect system, Autographa californica nuclear polyhidrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. A NGPCR gene coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofNGPCR gene coding sequence will result in inactivation of the polyhedringene and production of non-occluded recombinant virus (i.e., viruslacking the proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed (e.g., see Smith et al., 1983,J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0051] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NGPCR nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NGPCR gene product in infectedhosts (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NGPCR nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NGPCR gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NGPCR codingsequence is inserted, exogenous translational control signals,including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (SeeBittner et al., 1987, Methods in Enzymol. 153:516-544).

[0052] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, and WI38 cell lines.

[0053] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the NGPCR sequences described above may be engineered. Ratherthan using expression vectors that contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the NGPCR gene product. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds that affectthe endogenous activity of the NGPCR gene product.

[0054] A number of selection systems can be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes can be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

[0055] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺.nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

[0056] NGPCR gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate NGPCR transgenic animals.

[0057] Any technique known in the art may be used to introduce a NGPCRtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

[0058] The present invention provides for transgenic animals that carrythe NGPCR transgene in all their cells, as well as animals which carrythe transgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

[0059] When it is desired that a NGPCR transgene be integrated into thechromosomal site of the endogenous NGPCR gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NGPCRgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NGPCR gene (i.e.,“knockout” animals).

[0060] The transgene can also be selectively introduced into aparticular cell type, thus inactivating the endogenous NGPCR gene inonly that cell type, by following, for example, the teaching of Gu etal., 1994, Science, 265:103-106. The regulatory sequences required forsuch a cell-type specific inactivation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart.

[0061] Once transgenic animals have been generated, the expression ofthe recombinant NGPCR gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NGPCR gene-expressing tissue, may alsobe evaluated immunocytochemically using antibodies specific for theNGPCR transgene product.

5.3 Antibodies to NGPCR Proteins

[0062] Antibodies that specifically recognize one or more epitopes of aNGPCR, or epitopes of conserved variants of a NGPCR, or peptidefragments of a NGPCR are also encompassed by the invention. Suchantibodies include but are not limited to polyclonal antibodies,monoclonal antibodies (mAbs), humanized or chimeric antibodies, singlechain antibodies, Fab fragments, F(ab′)₂ fragments, fragments producedby a Fab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above.

[0063] The antibodies of the invention may be used, for example, in thedetection of NGPCR in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of NGPCR. Such antibodiesmay also be utilized in conjunction with, for example, compoundscreening schemes, as described below, for the evaluation of the effectof test compounds on expression and/or activity of a NGPCR gene product.Additionally, such antibodies can be used in conjunction gene therapyto, for example, evaluate the normal and/or engineered NGPCR-expressingcells prior to their introduction into the patient. Such antibodies mayadditionally be used as a method for the inhibition of abnormal NGPCRactivity. Thus, such antibodies may, therefore, be utilized as part ofweight disorder treatment methods.

[0064] For the production of antibodies, various host animals may beimmunized by injection with the NGPCR, an NGPCR peptide (e.g., onecorresponding the a functional domain of the receptor, such as an ECD,TM or CD), truncated NGPCR polypeptides (NGPCR in which one or moredomains, e.g., a TM or CD, has been deleted), functional equivalents ofthe NGPCR or mutants of the NGPCR. Such host animals may include but arenot limited to rabbits, mice, and rats, to name but a few. Variousadjuvants may be used to increase the immunological response, dependingon the host species, including but not limited to Freund's adjuvant(complete and incomplete), mineral salts such as aluminum hydroxide oraluminum phosphate, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Alternatively, the immune response could beenhanced by combination and or coupling with molecules such as keyholelimpet hemocyanin, tetanus toxoid, diptheria toxoid, ovalbumin, choleratoxin or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0065] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mabs in vivo makes this thepresently preferred method of production.

[0066] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures which are herein incorporatedby reference in their entirety.

[0067] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce single chain antibodies against NGPCR gene products. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0068] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0069] Antibodies to a NGPCR can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NGPCR, using techniqueswell known to those skilled in the art. (See, e.g., Greenspan & Bona,1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies which bind to a NGPCR ECD andcompetitively inhibit the binding of a ligand of NGPCR can be used togenerate anti-idiotypes that “mimic” a NGPCR ECD and, therefore, bindand neutralize a ligand. Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used in therapeutic regimensinvolving the NGPCR signaling pathway.

5.4 Diagnosis of Abnormalities Related to a NGPCR

[0070] A variety of methods can be employed for the diagnostic andprognostic evaluation of disorders related to NGPCR function, and forthe identification of subjects having a predisposition to suchdisorders.

[0071] Such methods can, for example, utilize reagents such as the NGPCRnucleotide sequences described in Section 5.1, and NGPCR antibodies, asdescribed, in Section 5.3. Specifically, such reagents may be used, forexample, for: (1) the detection of the presence of NGPCR gene mutations,or the detection of either over- or under-expression of NGPCR mRNArelative to a given phenotype; (2) the detection of either an over- oran under-abundance of NGPCR gene product relative to a given phenotype;and (3) the detection of perturbations or abnormalities in the signaltransduction pathway mediated by NGPCR.

[0072] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one specificNGPCR nucleotide sequence or NGPCR antibody reagent described herein,which may be conveniently used, e.g., in clinical settings, to diagnosepatients exhibiting body weight disorder abnormalities.

[0073] For the detection of NGPCR mutations, any nucleated cell can beused as a starting source for genomic nucleic acid. For the detection ofNGPCR gene expression or NGPCR gene products, any cell type or tissue inwhich the NGPCR gene is expressed, such as, for example, stomach orbrain cells can be utilized.

[0074] Nucleic acid-based detection techniques and peptide detectiontechniques are described below.

5.4.1 Detection of NGPCR Genes and Transcripts

[0075] Mutations within a NGPCR gene can be detected by utilizing anumber of techniques. Nucleic acid from any nucleated cell can be usedas the starting point for such assay techniques, and may be isolatedaccording to standard nucleic acid preparation procedures which are wellknown to those of skill in the art.

[0076] DNA may be used in hybridization or amplification assays ofbiological samples to detect abnormalities involving NGPCR genestructure, including point mutations, insertions, deletions andchromosomal rearrangements. Such assays may include, but are not limitedto, Southern analyses, single stranded conformational polymorphismanalyses (SSCP), and PCR analyses.

[0077] Such diagnostic methods for the detection of NGPCR gene-specificmutations can involve for example, contacting and incubating nucleicacids including recombinant DNA molecules, cloned genes or degeneratevariants thereof, obtained from a sample, e.g., derived from a patientsample or other appropriate cellular source, with one or more labelednucleic acid reagents including recombinant DNA molecules, cloned genesor degenerate variants thereof, as described in Section 5.1, underconditions favorable for the specific annealing of these reagents totheir complementary sequences within a given NGPCR gene. Preferably, thelengths of these nucleic acid reagents are at least 15 to 30nucleotides. After incubation, all non-annealed nucleic acids areremoved from the nucleic acid:NGPCR molecule hybrid. The presence ofnucleic acids which have hybridized, if any such molecules exist, isthen detected. Using such a detection scheme, the nucleic acid from thecell type or tissue of interest can be immobilized, for example, to asolid support such as a membrane, or a plastic surface such as that on amicrotiter plate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents of the type described inSection 5.1 are easily removed. Detection of the remaining, annealed,labeled NGPCR nucleic acid reagents is accomplished using standardtechniques well-known to those in the art. The NGPCR gene sequences towhich the nucleic acid reagents have annealed can be compared to theannealing pattern expected from a normal NGPCR gene sequence in order todetermine whether a NGPCR gene mutation is present.

[0078] Alternative diagnostic methods for the detection of NGPCR genespecific nucleic acid molecules, in patient samples or other appropriatecell sources, may involve their amplification, e.g., by PCR (theexperimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat. No.4,683,202), followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. The resultingamplified sequences can be compared to those which would be expected ifthe nucleic acid being amplified contained only normal copies of a NGPCRgene in order to determine whether a NGPCR gene mutation exists.

[0079] Additionally, well-known genotyping techniques can be performedto identify individuals carrying NGPCR gene mutations. Such techniquesinclude, for example, the use of restriction fragment lengthpolymorphisms (RFLPs), which involve sequence variations in one of therecognition sites for the specific restriction enzyme used.

[0080] Additionally, improved methods for analyzing DNA polymorphismswhich can be utilized for the identification of NGPCR gene mutationshave been described which capitalize on the presence of variable numbersof short, tandemly repeated DNA sequences between the restriction enzymesites. For example, Weber (U.S. Pat. No. 5,075,217, which isincorporated herein by reference in its entirety) describes a DNA markerbased on length polymorphisms in blocks of (dC-dA)n-(dG-dT)n shorttandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocks isestimated to be 30,000-60,000 bp. Markers which are so closely spacedexhibit a high frequency co-inheritance, and are extremely useful in theidentification of genetic mutations, such as, for example, mutationswithin a given NGPCR gene, and the diagnosis of diseases and disordersrelated to NGPCR mutations.

[0081] Also, Caskey et al. (U.S. Pat. No. 5,364,759, which isincorporated herein by reference in its entirety) describe a DNAprofiling assay for detecting short tri and tetra nucleotide repeatsequences. The process includes extracting the DNA of interest, such asthe NGPCR gene, amplifying the extracted DNA, and labeling the repeatsequences to form a genotypic map of the individual's DNA.

[0082] The level of NGPCR gene expression can also be assayed bydetecting and measuring NGPCR transcription. For example, RNA from acell type or tissue known, or suspected to express the NGPCR gene, suchas brain, may be isolated and tested utilizing hybridization or PCRtechniques such as are described, above. The isolated cells can bederived from cell culture or from a patient. The analysis of cells takenfrom culture may be a necessary step in the assessment of cells to beused as part of a cell-based gene therapy technique or, alternatively,to test the effect of compounds on the expression of the NGPCR gene.Such analyses may reveal both quantitative and qualitative aspects ofthe expression pattern of the NGPCR gene, including activation orinactivation of NGPCR gene expression.

[0083] Additionally, an oligonucleotide or polynucleotide sequence firstdisclosed in at least a portion of one or more of the NGPCR sequences ofSEQ ID NOS: 1-53 can be used as a hybridization probe in conjunctionwith a solid support matrix/substrate (resins, beads, membranes,plastics, polymers, metal or metallized substrates, crystalline orpolycrystalline substrates, etc.). Of particular note are spatiallyaddressable arrays (i.e., gene chips, microtiter plates, etc.) ofoligonucleotides and polynucleotides, or corresponding oligopeptides andpolypeptides, wherein at least one of the biopolymers present on thespatially addressable array comprises an oligonucleotide orpolynucleotide sequence first disclosed in at least one of the NGPCRsequences of SEQ ID NOS: 1-53, or an amino acid sequence encodedthereby. Methods for attaching biopolymers to, or synthesizingbiopolymers on, solid support matrices, and conducting binding studiesthereon are disclosed in, inter alia, U.S. Pat. Nos. 5,700,637,5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326,5,424,186, and 4,689,405 the disclosures of which are hereinincorporated by reference in their entirety.

[0084] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-53 can be used to identify and characterize the temporal andtissue specific expression of a gene. These addressable arraysincorporate oligonucleotide sequences of sufficient length to confer therequired specificity, yet be within the limitations of the productiontechnology. The length of these probes is within a range of betweenabout 8 to about 2000 nucleotides. Preferably the probes consist of 60nucleotides and more preferably 25 nucleotides from the sequences firstdisclosed in SEQ ID NOS:1-53.

[0085] For example, a series of the described NGPCR oligonucleotidesequences, or the complements thereof, can be used in chip format torepresent all or a portion of the described NGPCR sequences. Theoligonucleotides, typically between about 16 to about 40 (or any wholenumber within the stated range) nucleotides in length can partiallyoverlap each other and/or the NGPCR sequence may be represented usingoligonucleotides that do not overlap. Accordingly, the described NGPCRpolynucleotide sequences shall typically comprise at least about two orthree distinct oligonucleotide sequences of at least about 8 nucleotidesin length that are each first disclosed in the described SequenceListing. Such oligonucleotide sequences can begin at any nucleotidepresent within a sequence in the Sequence Listing and proceed in eithera sense (5′-to-3′) orientation vis-a-vis the described sequence or in anantisense orientation.

[0086] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-53 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

[0087] Probes consisting of sequences first disclosed in SEQ ID NOS:1-53can also be used in the identification, selection and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the drugs intended target.These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0088] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-53 can be utilized in microarrays or other assay formats, toscreen collections of genetic material from patients who have aparticular medical condition. These investigations can also be carriedout using the sequences first disclosed in SEQ ID NOS:1-53 in silico andby comparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

[0089] Thus the sequences first disclosed in SEQ ID NOS:1-53 can be usedto identify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

[0090] Although the presently described NGPCRs have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the NGPCRs can uniquely be described using any of a wide variety ofadditional structural attributes, or combinations thereof. For example,a given NGPCR can be described by the net composition of the nucleotidespresent within a given region of the NGPCR in conjunction with thepresence of one or more specific oligonucleotide sequence(s) firstdisclosed in the NGPCR. Alternatively, a restriction map specifying therelative positions of restriction endonuclease digestion sites, orvarious palindromic or other specific oligonucleotide sequences can beused to structurally describe a given NGPCR. Such restriction maps,which are typically generated by widely available computer programs(e.g., the University of Wisconsin GCG sequence analysis package,SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich., etc.), canoptionally be used in conjunction with one or more discrete nucleotidesequence(s) present in the NGPCR that can be described by the relativeposition of the sequence relative to one or more additional sequence(s)or one or more restriction sites present in the NGPCR.

[0091] In one embodiment of such a detection scheme, cDNAs aresynthesized from the RNAs of interest (e.g., by reverse transcription ofthe RNA molecule into CDNA). A sequence within the cDNA is then used asthe template for a nucleic acid amplification reaction, such as a PCRamplification reaction, or the like. The nucleic acid reagents used assynthesis initiation reagents (e.g., primers) in the reversetranscription and nucleic acid amplification steps of this method arechosen from among the NGPCR nucleic acid reagents described. Thepreferred lengths of such nucleic acid reagents are at least 9-30nucleotides. For detection of the amplified product, the nucleic acidamplification may be performed using radioactively or non-radioactivelylabeled nucleotides. Alternatively, enough amplified product may be madesuch that the product may be visualized by standard ethidium bromidestaining, by utilizing any other suitable nucleic acid staining method,or by sequencing.

[0092] Additionally, it is possible to perform such NGPCR geneexpression assays “in situ”, i.e., directly upon tissue sections (fixedand/or frozen) of patient tissue obtained from biopsies or resections,such that no nucleic acid purification is necessary. Nucleic acidreagents such as those described above may be used as probes and/orprimers for such in situ procedures (See, for example, Nuovo, G.J.,1992, “PCR In Situ Hybridization: Protocols And Applications”, RavenPress, NY).

[0093] Alternatively, if a sufficient quantity of the appropriate cellscan be obtained, standard Northern analysis can be performed todetermine the level of NGPCR mRNA expression.

5.4.2 Detection of NGPCR Gene Products

[0094] Antibodies directed against wild type or mutant NGPCR geneproducts or conserved variants or peptide fragments thereof, which arediscussed above, may also be used as diagnostics and prognostics, asdescribed herein. Such diagnostic methods, may be used to detectabnormalities in the level of NGPCR gene expression, or abnormalities inthe structure and/or temporal, tissue, cellular, or subcellular locationof the NGPCR, and may be performed in vivo or in vitro, such as, forexample, on biopsy tissue.

[0095] For example, antibodies directed to epitopes of the NGPCR ECD canbe used in vivo to detect the pattern and level of expression of theNGPCR in the body. Such antibodies can be labeled, e.g., with aradio-opaque or other appropriate compound and injected into a subjectin order to visualize binding to the NGPCR expressed in the body usingmethods such as X-rays, CAT-scans, or MRI. Labeled antibody fragments,e.g., the Fab or single chain antibody comprising the smallest portionof the antigen binding region, are preferred for this purpose to promotecrossing the blood-brain barrier and permit labeling NGPCRs expressed inthe brain.

[0096] Additionally, any NGPCR fusion protein or NGPCR conjugatedprotein whose presence can be detected, can be administered. Forexample, NGPCR fusion or conjugated proteins labeled with a radio-opaqueor other appropriate compound can be administered and visualized invivo, as discussed, above for labeled antibodies. Further such NGPCRfusion proteins as AP-NGPCR on NGPCR-Ap fusion proteins can be utilizedfor in vitro diagnostic procedures.

[0097] Alternatively, immunoassays or fusion protein detection assays,as described above, can be utilized on biopsy and autopsy samples invitro to permit assessment of the expression pattern of the NGPCR. Suchassays are not confined to the use of antibodies that define a NGPCRECD, but can include the use of antibodies directed to epitopes of anyof the domains of a NGPCR, e.g., the ECD, the TM and/or CD. The use ofeach or all of these labeled antibodies will yield useful informationregarding translation and intracellular transport of the NGPCR to thecell surface, and can identify defects in processing.

[0098] The tissue or cell type to be analyzed will generally includethose which are known, or suspected, to express the NGPCR gene. Theprotein isolation methods employed herein may, for example, be such asthose described in Harlow and Lane (Harlow, E. and Lane, D., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, New York), which is incorporated herein by referencein its entirety. The isolated cells can be derived from cell culture orfrom a patient. The analysis of cells taken from culture may be anecessary step in the assessment of cells that could be used as part ofa cell-based gene therapy technique or, alternatively, to test theeffect of compounds on the expression of a NGPCR gene.

[0099] For example, antibodies, or fragments of antibodies, such asthose described, useful in the present invention may be used toquantitatively or qualitatively detect the presence of NGPCR geneproducts or conserved variants or peptide fragments thereof. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below, this Section) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Suchtechniques are especially preferred if such NGPCR gene products areexpressed on the cell surface.

[0100] The antibodies (or fragments thereof) or NGPCR fusion orconjugated proteins useful in the present invention may, additionally,be employed histologically, as in immunofluorescence, immunoelectronmicroscopy or non-immuno assays, for in situ detection of NGPCR geneproducts or conserved variants or peptide fragments thereof, or forNGPCR binding (in the case of labeled NGPCR ligand fusion protein).

[0101] In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibody orfusion protein of the present invention. The antibody (or fragment) orfusion protein is preferably applied by overlaying the labeled antibody(or fragment) onto a biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of a NGPCRgene product, or conserved variants or peptide fragments, or NGPCRbinding, but also its distribution in the examined tissue. Using thepresent invention, those of ordinary skill will readily perceive thatany of a wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve such in situ detection.

[0102] Immunoassays and non-immunoassays for NGPCR gene products orconserved variants or peptide fragments thereof will typically compriseincubating a sample, such as a biological fluid, a tissue extract,freshly harvested cells, or lysates of cells which have been incubatedin cell culture, in the presence of a detectably labeled antibodycapable of identifying NGPCR gene products or conserved variants orpeptide fragments thereof, and detecting the bound antibody by any of anumber of techniques well-known in the art.

[0103] The biological sample may be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support which is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled NGPCR antibody or NGPCR ligand fusion protein. The solid phasesupport may then be washed with the buffer a second time to removeunbound antibody or fusion protein. The amount of bound label on solidsupport may then be detected by conventional means.

[0104] By “solid phase support or carrier” is intended any supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material can have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0105] The binding activity of a given lot of NGPCR antibody or NGPCRligand fusion protein may be determined according to well known methods.Those skilled in the art will be able to determine operative and optimalassay conditions for each determination by employing routineexperimentation.

[0106] With respect to antibodies, one of the ways in which the NGPCRantibody can be detectably labeled is by linking the same to an enzymeand use in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E.,1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.),1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme that is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

[0107] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect NGPCR throughthe use of a radioimmunoassay (RIA) (see, for example, Weintraub, B.,Principles of Radioimmunoassays, Seventh Training Course on RadioligandAssay Techniques, The Endocrine Society, March, 1986, which isincorporated by reference herein). The radioactive isotope can bedetected by such means as the use of a gamma counter or a scintillationcounter or by autoradiography.

[0108] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0109] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0110] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0111] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

5.5 Screening Assays for Compounds that Modulate NGPCR Expression orActivity

[0112] The following assays are designed to identify compounds thatinteract with (e.g., bind to) NGPCRs (including, but not limited to anECD or CD of a NGPCR), compounds that interact with (e.g., bind to)intracellular proteins that interact with NGPCR (including but notlimited to the TM and CD of NGPCR), compounds that interfere with theinteraction of NGPCR with transmembrane or intracellular proteinsinvolved in NGPCR-mediated signal transduction, and to compounds whichmodulate the activity of NGPCR gene (i.e., modulate the level of NGPCRgene expression) or modulate the level of NGPCR. Assays may additionallybe utilized which identify compounds which bind to NGPCR gene regulatorysequences (e.g., promoter sequences) and which may modulate NGPCR geneexpression. See e.g., Platt, K. A., 1994, J. Biol. Chem.269:28558-28562, which is incorporated herein by reference in itsentirety.

[0113] The compounds that can be screened in accordance with theinvention include but are not limited to peptides, antibodies andfragments thereof, and other organic compounds (e.g., peptidomimetics)that bind to an ECD of a NGPCR and either mimic the activity triggeredby the natural ligand (i.e., agonists) or inhibit the activity triggeredby the natural ligand (i.e., antagonists); as well as peptides,antibodies or fragments thereof, and other organic compounds that mimicthe ECD of the NGPCR (or a portion thereof) and bind to and “neutralize”the natural ligand.

[0114] Such compounds may include, but are not limited to, peptides suchas, for example, soluble peptides, including but not limited to membersof random peptide libraries; (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghten, R. et al., 1991, Nature 354:84-86), andcombinatorial chemistry-derived molecular library made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limitedto members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

[0115] Other compounds which can be screened in accordance with theinvention include but are not limited to small organic molecules thatare able to cross the blood-brain barrier, gain entry into anappropriate cell (e.g., in the choroid plexus, the hypothalamus, etc.)and affect the expression of a NGPCR gene or some other gene involved inthe NGPCR signal transduction pathway (e.g., by interacting with theregulatory region or transcription factors involved in gene expression);or such compounds that affect the activity of the NGPCR (e.g., byinhibiting or enhancing the enzymatic activity of a CD) or the activityof some other intracellular factor involved in the NGPCR signaltransduction pathway.

[0116] Computer modeling and searching technologies permitidentification of compounds, or the improvement of already identifiedcompounds, that can modulate NGPCR expression or activity. Havingidentified such a compound or composition, the active sites or regionsare identified. Such active sites might typically be ligand bindingsites. The active site can be identified using methods known in the artincluding, for example, from the amino acid sequences of peptides, fromthe nucleotide sequences of nucleic acids, or from study of complexes ofthe relevant compound or composition with its natural ligand. In thelatter case, chemical or X-ray crystallographic methods can be used tofind the active site by finding where on the factor the complexed ligandis found.

[0117] Next, the three dimensional geometric structure of the activesite is determined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intra-molecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed ligand, natural or artificial, which may increase the accuracyof the active site structure determined.

[0118] If an incomplete or insufficiently accurate structure isdetermined, the methods of computer based numerical modeling can be usedto complete the structure or improve its accuracy. Any recognizedmodeling method may be used, including parameterized models specific toparticular biopolymers such as proteins or nucleic acids, moleculardynamics models based on computing molecular motions, statisticalmechanics models based on thermal ensembles, or combined models. Formost types of models, standard molecular force fields, representing theforces between constituent atoms and groups, are necessary, and can beselected from force fields known in physical chemistry. The incompleteor less accurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

[0119] Finally, having determined the structure of the active site,either experimentally, by modeling, or by a combination, candidatemodulating compounds can be identified by searching databases containingcompounds along with information on their molecular structure. Such asearch seeks compounds having structures that match the determinedactive site structure and that interact with the groups defining theactive site. Such a search can be manual, but is preferably computerassisted. These compounds found from this search are potential NGPCRmodulating compounds.

[0120] Alternatively, these methods can be used to identify improvedmodulating compounds from an already known modulating compound orligand. The composition of the known compound can be modified and thestructural effects of modification can be determined using theexperimental and computer modeling methods described above applied tothe new composition. The altered structure is then compared to theactive site structure of the compound to determine if an improved fit orinteraction results. In this manner systematic variations incomposition, such as by varying side groups, can be quickly evaluated toobtain modified modulating compounds or ligands of improved specificityor activity.

[0121] Further experimental and computer modeling methods useful toidentify modulating compounds based upon identification of the activesites of a NGPCR, and related transduction and transcription factorswill be apparent to those of skill in the art.

[0122] Examples of molecular modeling systems are the CHARMM and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

[0123] A number of articles review computer modeling of drugsinteractive with specific proteins, such as Rotivinen, et al., 1988,Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist 54-57 (Jun.16, 1988); McKinaly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol.29:111-122; Perry and Davies, OSAR: Quantitative Structure-ActivityRelationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989);Lewis and Dean, 1989 Proc. R. Soc. Lond. 236:125-140 and 141-162; and,with respect to a model receptor for nucleic acid components, Askew, etal., 1989, J. Am. Chem. Soc. 111:1082-1090. Other computer programs thatscreen and graphically depict chemicals are available from companiessuch as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga,Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Althoughthese are primarily designed for application to drugs specific toparticular proteins, they can be adapted to design of drugs specific toregions of DNA or RNA, once that region is identified.

[0124] Although described above with reference to design and generationof compounds which could alter binding, one could also screen librariesof known compounds, including natural products or synthetic chemicals,and biologically active materials, including proteins, for compoundswhich are inhibitors or activators.

[0125] Cell-based systems can also be used to identify compounds thatbind NGPCRs as well as assess the altered activity associated with suchbinding in living cells. One tool of particular interest for such assaysis green fluorescent protein which is described, inter alia, in U.S.Pat. No. 5,625,048, herein incorporated by reference. Cells that may beused in such cellular assays include, but are not limited to,leukocytes, or cell lines derived from leukocytes, lymphocytes, stemcells, including embryonic stem cells, and the like. In addition,expression host cells (e.g., B95 cells, COS cells, CHO cells, OMK cells,fibroblasts, Sf9 cells) genetically engineered to express a functionalNGPCR of interest and to respond to activation by the test, or natural,ligand, as measured by a chemical or phenotypic change, or induction ofanother host cell gene, can be used as an end point in the assay.

[0126] Compounds identified via assays such as those described hereinmay be useful, for example, in elaborating the biological function of aNGPCR gene product. Such compounds can be administered to a patient attherapeutically effective doses to treat any of a variety ofphysiological or mental disorders. A therapeutically effective doserefers to that amount of the compound sufficient to result in anyamelioration, impediment, prevention, or alteration of any biological orovert symptom.

[0127] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0128] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0129] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients. Thus, thecompounds and their physiologically acceptable salts and solvates may beformulated for administration by inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, parenteral,intracranial, intrathecal, or rectal administration.

[0130] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0131] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound.

[0132] For buccal administration the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0133] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0134] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0135] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0136] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0137] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

5.5.1 In vitro Screening Assays for Compounds that Bind to NGPCRs

[0138] In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) NGPCR (including, but not limitedto, a ECD or CD of NGPCR). Compounds identified may be useful, forexample, in modulating the activity of wild type and/or mutant NGPCRgene products; may be useful in elaborating the biological function ofthe NGPCR; may be utilized in screens for identifying compounds thatdisrupt normal NGPCR interactions; or may in themselves disrupt suchinteractions.

[0139] The principle of the assays used to identify compounds that bindto the NGPCR involves preparing a reaction mixture of the NGPCR and thetest compound under conditions and for a time sufficient to allow thetwo components to interact and bind, thus forming a complex which can beremoved and/or detected in the reaction mixture. The NGPCR species usedcan vary depending upon the goal of the screening assay. For example,where agonists of the natural ligand are sought, the full length NGPCR,or a soluble truncated NGPCR, e.g., in which the TM and/or CD is deletedfrom the molecule, a peptide corresponding to a ECD or a fusion proteincontaining one or more NGPCR ECD fused to a protein or polypeptide thataffords advantages in the assay system (e.g., labeling, isolation of theresulting complex, etc.) can be utilized. Where compounds that interactwith the cytoplasmic domain are sought to be identified, peptidescorresponding to the NGPCR CD and fusion proteins containing the NGPCRCD can be used.

[0140] The screening assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoring theNGPCR protein, polypeptide, peptide or fusion protein or the testsubstance onto a solid phase and detecting NGPCR/test compound complexesanchored on the solid phase at the end of the reaction. In oneembodiment of such a method, the NGPCR reactant may be anchored onto asolid surface, and the test compound, which is not anchored, may belabeled, either directly or indirectly.

[0141] In practice, microtiter plates may conveniently be utilized asthe solid phase. The anchored component may be immobilized bynon-covalent or covalent attachments. Non-covalent attachment may beaccomplished by simply coating the solid surface with a solution of theprotein and drying. Alternatively, an immobilized antibody, preferably amonoclonal antibody, specific for the protein to be immobilized may beused to anchor the protein to the solid surface. The surfaces may beprepared in advance and stored.

[0142] In order to conduct the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously nonimmobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously nonimmobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the previouslynonimmobilized component (the antibody, in turn, may be directly labeledor indirectly labeled with a labeled anti-Ig antibody).

[0143] Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for a NGPCRprotein, polypeptide, peptide or fusion protein or the test compound toanchor any complexes formed in solution, and a labeled antibody specificfor the other component of the possible complex to detect anchoredcomplexes.

[0144] Alternatively, cell-based assays can be used to identifycompounds that interact with NGPCR. To this end, cell lines that expressNGPCR, or cell lines (e.g., COS cells, CHO cells, fibroblasts, etc.)that have been genetically engineered to express a NGPCR (e.g., bytransfection or transduction of NGPCR DNA) can be used. Interaction ofthe test compound with, for example, a ECD of a NGPCR expressed by thehost cell can be determined by comparison or competition with nativeligand.

5.5.2. Assays for Intracellular Proteins that Interact with NGPCRs

[0145] Any method suitable for detecting protein-protein interactionsmay be employed for identifying transmembrane proteins or intracellularproteins that interact with a NGPCR. Among the traditional methods whichmay be employed are co-immunoprecipitation, crosslinking andco-purification through gradients or chromatographic columns of celllysates or proteins obtained from cell lysates and a NGPCR to identifyproteins in the lysate that interact with the NGPCR. For these assays,the NGPCR component used can be a full length NGPCR, a solublederivative lacking the membrane-anchoring region (e.g., a truncatedNGPCR in which a TM is deleted resulting in a truncated moleculecontaining a ECD fused to a CD), a peptide corresponding to a CD or afusion protein containing a CD of a NGPCR. Once isolated, such anintracellular protein can be identified and can, in turn, be used, inconjunction with standard techniques, to identify proteins with which itinteracts. For example, at least a portion of the amino acid sequence ofan intracellular protein which interacts with a NGPCR can be ascertainedusing techniques well known to those of skill in the art, such as viathe Edman degradation technique. (See, e.g., Creighton, 1983, “Proteins:Structures and Molecular Principles”, W.H. Freeman & Co., N.Y.,pp.34-49). The amino acid sequence obtained may be used as a guide forthe generation of oligonucleotide mixtures that can be used to screenfor gene sequences encoding such intracellular proteins. Screening canbe accomplished, for example, by standard hybridization or PCRtechniques. Techniques for the generation of oligonucleotide mixturesand the screening are well-known. (See, e.g., Ausubel, supra, and PCRProtocols: A Guide to Methods and Applications, 1990, Innis, M. et al.,eds. Academic Press, Inc., New York).

[0146] Additionally, methods may be employed which result in thesimultaneous identification of genes which encode the transmembrane orintracellular proteins interacting with NGPCR. These methods include,for example, probing expression, libraries, in a manner similar to thewell known technique of antibody probing of λgt11 libraries, usinglabeled NGPCR protein, or an NGPCR polypeptide, peptide or fusionprotein, e.g., an NGPCR polypeptide or NGPCR domain fused to a marker(e.g., an enzyme, fluor, luminescent protein, or dye), or an Ig-Fcdomain.

[0147] One method that detects protein interactions in vivo, thetwo-hybrid system, is described in detail for illustration only and notby way of limitation. One version of this system has been described(Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.).

[0148] Briefly, utilizing such a system, plasmids are constructed thatencode two hybrid proteins: one plasmid consists of nucleotides encodingthe DNA-binding domain of a transcription activator protein fused to aNGPCR nucleotide sequence encoding NGPCR, an NGPCR polypeptide, peptideor fusion protein, and the other plasmid consists of nucleotidesencoding the transcription activator protein's activation domain fusedto a cDNA encoding an unknown protein which has been recombined intothis plasmid as part of a cDNA library. The DNA-binding domain fusionplasmid and the cDNA library are transformed into a strain of the yeastSaccharomyces cerevisiae that contains a reporter gene (e.g., HBS orlacZ) whose regulatory region contains the transcription activator'sbinding site. Either hybrid protein alone cannot activate transcriptionof the reporter gene: the DNA-binding domain hybrid cannot because itdoes not provide activation function and the activation domain hybridcannot because it cannot localize to the activator's binding sites.Interaction of the two hybrid proteins reconstitutes the functionalactivator protein and results in expression of the reporter gene, whichis detected by an assay for the reporter gene product.

[0149] The two-hybrid system or related methodology may be used toscreen activation domain libraries for proteins that interact with the“bait” gene product. By way of example, and not by way of limitation, aNGPCR may be used as the bait gene product. Total genomic or cDNAsequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of a bait NGPCR gene productfused to the DNA-binding domain are cotransformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene. For example, and not by way of limitation, abait NGPCR gene sequence, such as the open reading frame of a NGPCR (ora domain of a NGPCR) can be cloned into a vector such that it istranslationally fused to the DNA encoding the DNA-binding domain of theGAL4 protein. These colonies are purified and the library plasmidsresponsible for reporter gene expression are isolated. DNA sequencing isthen used to identify the proteins encoded by the library plasmids.

[0150] A cDNA library of the cell line from which proteins that interactwith bait NGPCR gene product are to be detected can be made usingmethods routinely practiced in the art. According to the particularsystem described herein, for example, the cDNA fragments can be insertedinto a vector such that they are translationally fused to thetranscriptional activation domain of GAL4. This library can beco-transformed along with the bait NGPCR gene-GAL4 fusion plasmid into ayeast strain which contains a lacZ gene driven by a promoter whichcontains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4transcriptional activation domain, that interacts with bait NGPCR geneproduct will reconstitute an active GAL4 protein and thereby driveexpression of the HIS3 gene. Colonies which express HIS3 can be detectedby their growth on petri dishes containing semi-solid agar based medialacking histidine. The cDNA can then be purified from these strains, andused to produce and isolate the bait NGPCR gene-interacting proteinusing techniques routinely practiced in the art.

5.5.3. Assays for Compounds that Interfere with NGPCR/Intracellular orNGPCR/Transmembrane Macromolecule Interaction

[0151] The macromolecules that interact with the NGPCR are referred to,for purposes of this discussion, as “binding partners.” These bindingpartners are likely to be involved in the NGPCR signal transductionpathway. Therefore, it is desirable to identify compounds that interferewith or disrupt the interaction of such binding partners which may beuseful in regulating the activity of a NGPCR and controlling disordersassociated with NGPCR activity. For example, given their expressionpattern, the described NGPCRs are contemplated to be particularly usefulin methods for identifying compounds useful in the therapeutic treatmentof obesity, inflammation, immune disorders, diabetes, heart and coronarydisease, metabolic disorders, and cancer.

[0152] The basic principle of the assay systems used to identifycompounds that interfere with the interaction between a NGPCR and itsbinding partner or partners involves preparing a reaction mixturecontaining NGPCR protein, polypeptide, peptide or fusion protein asdescribed, and the binding partner under conditions and for a timesufficient to allow the two to interact and bind, thus forming acomplex. In order to test a compound for inhibitory activity, thereaction mixture is prepared in the presence and absence of the testcompound. The test compound may be initially included in the reactionmixture, or may be added at a time subsequent to the addition of theNGPCR moiety and its binding partner. Control reaction mixtures areincubated without the test compound or with a placebo. The formation ofany complexes between the NGPCR moiety and the binding partner is thendetected. The formation of a complex in the control reaction, but not inthe reaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the NGPCR and theinteractive binding partner. Additionally, complex formation withinreaction mixtures containing the test compound and normal NGPCR proteinmay also be compared to complex formation within reaction mixturescontaining the test compound and a mutant NGPCR. This comparison may beimportant in those cases wherein it is desirable to identify compoundsthat specifically disrupt interactions of mutant, or mutated, NGPCRs butnot normal NGPCRs.

[0153] The assay for compounds that interfere with the interaction of aNGPCR and its binding partners can be conducted in a heterogeneous orhomogeneous format. Heterogeneous assays involve anchoring either theNGPCR moiety product or the binding partner onto a solid phase anddetecting complexes anchored on the solid phase at the end of thereaction. In homogeneous assays, the entire reaction is carried out in aliquid phase. In either approach, the order of addition of reactants canbe varied to obtain different information about the compounds beingtested. For example, test compounds that interfere with the interactionby competition can be identified by conducting the reaction in thepresence of the test substance; i.e., by adding the test substance tothe reaction mixture prior to, or simultaneously with, a NGPCR moietyand interactive binding partner. Alternatively, test compounds thatdisrupt preformed complexes, e.g. compounds with higher bindingconstants that displace one of the components from the complex, can betested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are described brieflybelow.

[0154] In a heterogeneous assay system, either a NGPCR moiety or aninteractive binding partner, is anchored onto a solid surface, while thenon-anchored species is labeled, either directly or indirectly. Inpractice, microtiter plates are conveniently utilized. The anchoredspecies may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of the NGPCR gene product or binding partner anddrying. Alternatively, an immobilized antibody specific for the speciesto be anchored may be used to anchor the species to the solid surface.The surfaces may be prepared in advance and stored.

[0155] In order to conduct the assay, the partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thenon-immobilized species is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe non-immobilized species is not pre-labeled, an indirect label can beused to detect complexes anchored on the surface; e.g., using a labeledantibody specific for the initially non-immobilized species (theantibody, in turn, may be directly labeled or indirectly labeled with alabeled anti-Ig antibody). Depending upon the order of addition ofreaction components, test compounds which inhibit complex formation orwhich disrupt preformed complexes can be detected.

[0156] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

[0157] In an alternate embodiment of the invention, a homogeneous assaycan be used. In this approach, a preformed complex of a NGPCR moiety andan interactive binding partner is prepared in which either the NGPCR orits binding partners is labeled, but the signal generated by the labelis quenched due to formation of the complex (see, e.g., U.S. Pat. No.4,109,496 by Rubenstein which utilizes this approach for immunoassays).The addition of a test substance that competes with and displaces one ofthe species from the preformed complex will result in the generation ofa signal above background. In this way, test substances which disruptNGPCR/intracellular binding partner interaction can be identified.

[0158] In a particular embodiment, a NGPCR fusion can be prepared forimmobilization. For example, a NGPCR or a peptide fragment, e.g.,corresponding to a CD, can be fused to a glutathione-S-transferase (GST)gene using a fusion vector, such as pGEX-5X-1, in such a manner that itsbinding activity is maintained in the resulting fusion protein. Theinteractive binding partner can be purified and used to raise amonoclonal antibody, using methods routinely practiced in the art anddescribed above, in Section 5.3. This antibody can be labeled with theradioactive isotope ¹²⁵I, for example, by methods routinely practiced inthe art. In a heterogeneous assay, e.g., the GST-NGPCR fusion proteincan be anchored to glutathione-agarose beads. The interactive bindingpartner can then be added in the presence or absence of the testcompound in a manner that allows interaction and binding to occur. Atthe end of the reaction period, unbound material can be washed away, andthe labeled monoclonal antibody can be added to the system and allowedto bind to the complexed components. The interaction between a NGPCRgene product and the interactive binding partner can be detected bymeasuring the amount of radioactivity that remains associated with theglutathione-agarose beads. A successful inhibition of the interaction bythe test compound will result in a decrease in measured radioactivity.

[0159] Alternatively, the GST-NGPCR fusion protein and the interactivebinding partner can be mixed together in liquid in the absence of thesolid glutathione-agarose beads. The test compound can be added eitherduring or after the species are allowed to interact. This mixture canthen be added to the glutathione-agarose beads and unbound material iswashed away. Again the extent of inhibition of the NGPCR/binding partnerinteraction can be detected by adding the labeled antibody and measuringthe radioactivity associated with the beads.

[0160] In another embodiment of the invention, these same techniques canbe employed using peptide fragments that correspond to the bindingdomains of a NGPCR and/or the interactive or binding partner (in caseswhere the binding partner is a protein), in place of one or both of thefull length proteins. Any number of methods routinely practiced in theart can be used to identify and isolate the binding sites. These methodsinclude, but are not limited to, mutagenesis of the gene encoding one ofthe proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensatory mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, arelatively short, labeled peptide comprising the binding domain mayremain associated with the solid material, which can be isolated andidentified by amino acid sequencing. Also, once the gene coding for theintracellular binding partner is obtained, short gene segments can beengineered to express peptide fragments of the protein, which can thenbe tested for binding activity and purified or synthesized.

[0161] For example, and not by way of limitation, a NGPCR gene productcan be anchored to a solid material as described, above, by making aGST-NGPCR fusion protein and allowing it to bind to glutathione agarosebeads. The interactive binding partner can be labeled with a radioactiveisotope, such as ³⁵S, and cleaved with a proteolytic enzyme such astrypsin. Cleavage products can then be added to the anchored GST-NGPCRfusion protein and allowed to bind. After washing away unbound peptides,labeled bound material, representing the intracellular binding partnerbinding domain, can be eluted, purified, and analyzed for amino acidsequence by well-known methods. Peptides so identified can be producedsynthetically or fused to appropriate facilitative proteins usingrecombinant DNA technology.

[0162] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims. All referenced publications, patents, and patent applicationsare herein incorporated by reference.

1 53 1 678 DNA Homo sapiens 1 atggcgacgc ccaggggcct gggggccctgctcctgctcc tcctgctccc gacctcaggt 60 caggaaaagc ccaccgaagg gccaagaaacacctgcctgg ggagcaacaa catgtacgac 120 atcttcaact tgaatgacaa ggctttgtgcttcaccaagt gcaggcagtc gggcagcgac 180 tcctgcaatg tggaaaactt gcagagatactggctaaact acgaggccca tctgatgaag 240 gaaggtttga cgcagaaggt gaacacgcctttcctgaagg ctttggtcca gaacctcagc 300 accaacactg cagaagactt ctatttctctctggagccct ctcaggttcc gaggcaggtg 360 atgaaggacg aggacaagcc ccctgacagagtgcgacttc ccaagagcct ttttcgatcc 420 ctgccaggca acaggtctgt ggtccgcttggccgtcacca ttctggacat tggtccaggg 480 actctcttca agggcccccg gctcggcctgggagatggca gcggcgtgtt gaacaatcgc 540 ctggtgggtt tgagtgtggg acaaatgcatgtcaccaagc tggctgagcc tctggagatc 600 gtcttctctc accagcgacc gccccctgtgagtcccctgc tcaggcctgg cagccactgc 660 agggcagaca gaacatga 678 2 225 PRTHomo sapiens 2 Met Ala Thr Pro Arg Gly Leu Gly Ala Leu Leu Leu Leu LeuLeu Leu 1 5 10 15 Pro Thr Ser Gly Gln Glu Lys Pro Thr Glu Gly Pro ArgAsn Thr Cys 20 25 30 Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu AsnAsp Lys Ala 35 40 45 Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp SerCys Asn Val 50 55 60 Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala HisLeu Met Lys 65 70 75 80 Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe LeuLys Ala Leu Val 85 90 95 Gln Asn Leu Ser Thr Asn Thr Ala Glu Asp Phe TyrPhe Ser Leu Glu 100 105 110 Pro Ser Gln Val Pro Arg Gln Val Met Lys AspGlu Asp Lys Pro Pro 115 120 125 Asp Arg Val Arg Leu Pro Lys Ser Leu PheArg Ser Leu Pro Gly Asn 130 135 140 Arg Ser Val Val Arg Leu Ala Val ThrIle Leu Asp Ile Gly Pro Gly 145 150 155 160 Thr Leu Phe Lys Gly Pro ArgLeu Gly Leu Gly Asp Gly Ser Gly Val 165 170 175 Leu Asn Asn Arg Leu ValGly Leu Ser Val Gly Gln Met His Val Thr 180 185 190 Lys Leu Ala Glu ProLeu Glu Ile Val Phe Ser His Gln Arg Pro Pro 195 200 205 Pro Val Ser ProLeu Leu Arg Pro Gly Ser His Cys Arg Ala Asp Arg 210 215 220 Thr 225 31527 DNA Homo sapiens 3 atggcgacgc ccaggggcct gggggccctg ctcctgctcctcctgctccc gacctcaggt 60 caggaaaagc ccaccgaagg gccaagaaac acctgcctggggagcaacaa catgtacgac 120 atcttcaact tgaatgacaa ggctttgtgc ttcaccaagtgcaggcagtc gggcagcgac 180 tcctgcaatg tggaaaactt gcagagatac tggctaaactacgaggccca tctgatgaag 240 gaaggtttga cgcagaaggt gaacacgcct ttcctgaaggctttggtcca gaacctcagc 300 accaacactg cagaagactt ctatttctct ctggagccctctcaggttcc gaggcaggtg 360 atgaaggacg aggacaagcc ccctgacaga gtgcgacttcccaagagcct ttttcgatcc 420 ctgccaggca acaggtctgt ggtccgcttg gccgtcaccattctggacat tggtccaggg 480 actctcttca agggcccccg gctcggcctg ggagatggcagcggcgtgtt gaacaatcgc 540 ctggtgggtt tgagtgtggg acaaatgcat gtcaccaagctggctgagcc tctggagatc 600 gtcttctctc accagcgacc gccccctaac atgaccctcacctgtgtatt ctgggatgtg 660 actaaaggga ccactggaga ctggtcttct gagggctgctccacggaggt cagacctgag 720 gggaccgtgt gctgctgtga ccacctgacc tttttcgccctgctcctgag acccaccttg 780 gaccagtcca cggtgcatat cctcacacgc atctcccaggcgggctgtgg ggtctccatg 840 atcttcctgg ccttcaccat tattctttat gcctttctgaggctttcccg ggagaggttc 900 aagtcagaag atgccccaaa gatccacgtg gccctgggtggcagcctgtt cctcctgaat 960 ctggccttct tggtcaatgt ggggagtggc tcaaaggggtctgatgctgc ctgctgggcc 1020 cggggggctg tcttccacta cttcctgctc tgtgccttcacctggatggg ccttgaagcc 1080 ttccacctct acctgctcgc tgtcagggtc ttcaacacctacttcgggca ctacttcctg 1140 aagctgagcc tggtgggctg gggcctgccc gccctgatggtcatcggcac tgggagtgcc 1200 aacagctacg gcctctacac catccgtgat agggagaaccgcacctctct ggagctatgc 1260 tggttccgtg aagggacaac catgtacgcc ctctatatcaccgtccacgg ctacttcctc 1320 atcaccttcc tctttggcat ggtggtcctg gccctggtggtctggaagat cttcaccctg 1380 tcccgtgcta cagcggtcaa ggagcggggg aagaaccggaagaaggtgct caccctgctg 1440 ggcctctcga gccttgcaag ttgggtgtcc atcgtccatctctggtccaa tcagctgcga 1500 ccagaagggc agaatcatgt gatatga 1527 4 508 PRTHomo sapiens 4 Met Ala Thr Pro Arg Gly Leu Gly Ala Leu Leu Leu Leu LeuLeu Leu 1 5 10 15 Pro Thr Ser Gly Gln Glu Lys Pro Thr Glu Gly Pro ArgAsn Thr Cys 20 25 30 Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu AsnAsp Lys Ala 35 40 45 Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp SerCys Asn Val 50 55 60 Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala HisLeu Met Lys 65 70 75 80 Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe LeuLys Ala Leu Val 85 90 95 Gln Asn Leu Ser Thr Asn Thr Ala Glu Asp Phe TyrPhe Ser Leu Glu 100 105 110 Pro Ser Gln Val Pro Arg Gln Val Met Lys AspGlu Asp Lys Pro Pro 115 120 125 Asp Arg Val Arg Leu Pro Lys Ser Leu PheArg Ser Leu Pro Gly Asn 130 135 140 Arg Ser Val Val Arg Leu Ala Val ThrIle Leu Asp Ile Gly Pro Gly 145 150 155 160 Thr Leu Phe Lys Gly Pro ArgLeu Gly Leu Gly Asp Gly Ser Gly Val 165 170 175 Leu Asn Asn Arg Leu ValGly Leu Ser Val Gly Gln Met His Val Thr 180 185 190 Lys Leu Ala Glu ProLeu Glu Ile Val Phe Ser His Gln Arg Pro Pro 195 200 205 Pro Asn Met ThrLeu Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr 210 215 220 Thr Gly AspTrp Ser Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu 225 230 235 240 GlyThr Val Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu 245 250 255Arg Pro Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser 260 265270 Gln Ala Gly Cys Gly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile 275280 285 Leu Tyr Ala Phe Leu Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp290 295 300 Ala Pro Lys Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu LeuAsn 305 310 315 320 Leu Ala Phe Leu Val Asn Val Gly Ser Gly Ser Lys GlySer Asp Ala 325 330 335 Ala Cys Trp Ala Arg Gly Ala Val Phe His Tyr PheLeu Leu Cys Ala 340 345 350 Phe Thr Trp Met Gly Leu Glu Ala Phe His LeuTyr Leu Leu Ala Val 355 360 365 Arg Val Phe Asn Thr Tyr Phe Gly His TyrPhe Leu Lys Leu Ser Leu 370 375 380 Val Gly Trp Gly Leu Pro Ala Leu MetVal Ile Gly Thr Gly Ser Ala 385 390 395 400 Asn Ser Tyr Gly Leu Tyr ThrIle Arg Asp Arg Glu Asn Arg Thr Ser 405 410 415 Leu Glu Leu Cys Trp PheArg Glu Gly Thr Thr Met Tyr Ala Leu Tyr 420 425 430 Ile Thr Val His GlyTyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val 435 440 445 Val Leu Ala LeuVal Val Trp Lys Ile Phe Thr Leu Ser Arg Ala Thr 450 455 460 Ala Val LysGlu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr Leu Leu 465 470 475 480 GlyLeu Ser Ser Leu Ala Ser Trp Val Ser Ile Val His Leu Trp Ser 485 490 495Asn Gln Leu Arg Pro Glu Gly Gln Asn His Val Ile 500 505 5 897 DNA Homosapiens 5 atgaccctca cctgtgtatt ctgggatgtg actaaaggga ccactggagactggtcttct 60 gagggctgct ccacggaggt cagacctgag gggaccgtgt gctgctgtgaccacctgacc 120 tttttcgccc tgctcctgag acccaccttg gaccagtcca cggtgcatatcctcacacgc 180 atctcccagg cgggctgtgg ggtctccatg atcttcctgg ccttcaccattattctttat 240 gcctttctga ggctttcccg ggagaggttc aagtcagaag atgccccaaagatccacgtg 300 gccctgggtg gcagcctgtt cctcctgaat ctggccttct tggtcaatgtggggagtggc 360 tcaaaggggt ctgatgctgc ctgctgggcc cggggggctg tcttccactacttcctgctc 420 tgtgccttca cctggatggg ccttgaagcc ttccacctct acctgctcgctgtcagggtc 480 ttcaacacct acttcgggca ctacttcctg aagctgagcc tggtgggctggggcctgccc 540 gccctgatgg tcatcggcac tgggagtgcc aacagctacg gcctctacaccatccgtgat 600 agggagaacc gcacctctct ggagctatgc tggttccgtg aagggacaaccatgtacgcc 660 ctctatatca ccgtccacgg ctacttcctc atcaccttcc tctttggcatggtggtcctg 720 gccctggtgg tctggaagat cttcaccctg tcccgtgcta cagcggtcaaggagcggggg 780 aagaaccgga agaaggtgct caccctgctg ggcctctcga gccttgcaagttgggtgtcc 840 atcgtccatc tctggtccaa tcagctgcga ccagaagggc agaatcatgtgatatga 897 6 298 PRT Homo sapiens 6 Met Thr Leu Thr Cys Val Phe Trp AspVal Thr Lys Gly Thr Thr Gly 1 5 10 15 Asp Trp Ser Ser Glu Gly Cys SerThr Glu Val Arg Pro Glu Gly Thr 20 25 30 Val Cys Cys Cys Asp His Leu ThrPhe Phe Ala Leu Leu Leu Arg Pro 35 40 45 Thr Leu Asp Gln Ser Thr Val HisIle Leu Thr Arg Ile Ser Gln Ala 50 55 60 Gly Cys Gly Val Ser Met Ile PheLeu Ala Phe Thr Ile Ile Leu Tyr 65 70 75 80 Ala Phe Leu Arg Leu Ser ArgGlu Arg Phe Lys Ser Glu Asp Ala Pro 85 90 95 Lys Ile His Val Ala Leu GlyGly Ser Leu Phe Leu Leu Asn Leu Ala 100 105 110 Phe Leu Val Asn Val GlySer Gly Ser Lys Gly Ser Asp Ala Ala Cys 115 120 125 Trp Ala Arg Gly AlaVal Phe His Tyr Phe Leu Leu Cys Ala Phe Thr 130 135 140 Trp Met Gly LeuGlu Ala Phe His Leu Tyr Leu Leu Ala Val Arg Val 145 150 155 160 Phe AsnThr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu Val Gly 165 170 175 TrpGly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser 180 185 190Tyr Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu 195 200205 Leu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr 210215 220 Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val Val Leu225 230 235 240 Ala Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg Ala ThrAla Val 245 250 255 Lys Glu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr LeuLeu Gly Leu 260 265 270 Ser Ser Leu Ala Ser Trp Val Ser Ile Val His LeuTrp Ser Asn Gln 275 280 285 Leu Arg Pro Glu Gly Gln Asn His Val Ile 290295 7 1080 DNA Homo sapiens 7 atggcccctt ctgcagcctg gcctccccgatctccccttt cacagggccc ccggctcggc 60 ctgggagatg gcagcggcgt gttgaacaatcgcctggtgg gtttgagtgt gggacaaatg 120 catgtcacca agctggctga gcctctggagatcgtcttct ctcaccagcg accgccccct 180 aacatgaccc tcacctgtgt attctgggatgtgactaaag ggaccactgg agactggtct 240 tctgagggct gctccacgga ggtcagacctgaggggaccg tgtgctgctg tgaccacctg 300 acctttttcg ccctgctcct gagacccaccttggaccagt ccacggtgca tatcctcaca 360 cgcatctccc aggcgggctg tggggtctccatgatcttcc tggccttcac cattattctt 420 tatgcctttc tgaggctttc ccgggagaggttcaagtcag aagatgcccc aaagatccac 480 gtggccctgg gtggcagcct gttcctcctgaatctggcct tcttggtcaa tgtggggagt 540 ggctcaaagg ggtctgatgc tgcctgctgggcccgggggg ctgtcttcca ctacttcctg 600 ctctgtgcct tcacctggat gggccttgaagccttccacc tctacctgct cgctgtcagg 660 gtcttcaaca cctacttcgg gcactacttcctgaagctga gcctggtggg ctggggcctg 720 cccgccctga tggtcatcgg cactgggagtgccaacagct acggcctcta caccatccgt 780 gatagggaga accgcacctc tctggagctatgctggttcc gtgaagggac aaccatgtac 840 gccctctata tcaccgtcca cggctacttcctcatcacct tcctctttgg catggtggtc 900 ctggccctgg tggtctggaa gatcttcaccctgtcccgtg ctacagcggt caaggagcgg 960 gggaagaacc ggaagaaggt gctcaccctgctgggcctct cgagccttgc aagttgggtg 1020 tccatcgtcc atctctggtc caatcagctgcgaccagaag ggcagaatca tgtgatatga 1080 8 359 PRT Homo sapiens 8 Met AlaPro Ser Ala Ala Trp Pro Pro Arg Ser Pro Leu Ser Gln Gly 1 5 10 15 ProArg Leu Gly Leu Gly Asp Gly Ser Gly Val Leu Asn Asn Arg Leu 20 25 30 ValGly Leu Ser Val Gly Gln Met His Val Thr Lys Leu Ala Glu Pro 35 40 45 LeuGlu Ile Val Phe Ser His Gln Arg Pro Pro Pro Asn Met Thr Leu 50 55 60 ThrCys Val Phe Trp Asp Val Thr Lys Gly Thr Thr Gly Asp Trp Ser 65 70 75 80Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu Gly Thr Val Cys Cys 85 90 95Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu Arg Pro Thr Leu Asp 100 105110 Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser Gln Ala Gly Cys Gly 115120 125 Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile Leu Tyr Ala Phe Leu130 135 140 Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp Ala Pro Lys IleHis 145 150 155 160 Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn Leu AlaPhe Leu Val 165 170 175 Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala AlaCys Trp Ala Arg 180 185 190 Gly Ala Val Phe His Tyr Phe Leu Leu Cys AlaPhe Thr Trp Met Gly 195 200 205 Leu Glu Ala Phe His Leu Tyr Leu Leu AlaVal Arg Val Phe Asn Thr 210 215 220 Tyr Phe Gly His Tyr Phe Leu Lys LeuSer Leu Val Gly Trp Gly Leu 225 230 235 240 Pro Ala Leu Met Val Ile GlyThr Gly Ser Ala Asn Ser Tyr Gly Leu 245 250 255 Tyr Thr Ile Arg Asp ArgGlu Asn Arg Thr Ser Leu Glu Leu Cys Trp 260 265 270 Phe Arg Glu Gly ThrThr Met Tyr Ala Leu Tyr Ile Thr Val His Gly 275 280 285 Tyr Phe Leu IleThr Phe Leu Phe Gly Met Val Val Leu Ala Leu Val 290 295 300 Val Trp LysIle Phe Thr Leu Ser Arg Ala Thr Ala Val Lys Glu Arg 305 310 315 320 GlyLys Asn Arg Lys Lys Val Leu Thr Leu Leu Gly Leu Ser Ser Leu 325 330 335Ala Ser Trp Val Ser Ile Val His Leu Trp Ser Asn Gln Leu Arg Pro 340 345350 Glu Gly Gln Asn His Val Ile 355 9 702 DNA Homo sapiens 9 atgggagctccccatgggag ctgtggcccc ttggggcctc ttatttctca ccccaggctt 60 tcccgggagaggttcaagtc agaagatgcc ccaaagatcc acgtggccct gggtggcagc 120 ctgttcctcctgaatctggc cttcttggtc aatgtgggga gtggctcaaa ggggtctgat 180 gctgcctgctgggcccgggg ggctgtcttc cactacttcc tgctctgtgc cttcacctgg 240 atgggccttgaagccttcca cctctacctg ctcgctgtca gggtcttcaa cacctacttc 300 gggcactacttcctgaagct gagcctggtg ggctggggcc tgcccgccct gatggtcatc 360 ggcactgggagtgccaacag ctacggcctc tacaccatcc gtgataggga gaaccgcacc 420 tctctggagctatgctggtt ccgtgaaggg acaaccatgt acgccctcta tatcaccgtc 480 cacggctacttcctcatcac cttcctcttt ggcatggtgg tcctggccct ggtggtctgg 540 aagatcttcaccctgtcccg tgctacagcg gtcaaggagc gggggaagaa ccggaagaag 600 gtgctcaccctgctgggcct ctcgagcctt gcaagttggg tgtccatcgt ccatctctgg 660 tccaatcagctgcgaccaga agggcagaat catgtgatat ga 702 10 233 PRT Homo sapiens 10 MetGly Ala Pro His Gly Ser Cys Gly Pro Leu Gly Pro Leu Ile Ser 1 5 10 15His Pro Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp Ala Pro Lys 20 25 30Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn Leu Ala Phe 35 40 45Leu Val Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala Ala Cys Trp 50 55 60Ala Arg Gly Ala Val Phe His Tyr Phe Leu Leu Cys Ala Phe Thr Trp 65 70 7580 Met Gly Leu Glu Ala Phe His Leu Tyr Leu Leu Ala Val Arg Val Phe 85 9095 Asn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu Val Gly Trp 100105 110 Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser Tyr115 120 125 Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu GluLeu 130 135 140 Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr IleThr Val 145 150 155 160 His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly MetVal Val Leu Ala 165 170 175 Leu Val Val Trp Lys Ile Phe Thr Leu Ser ArgAla Thr Ala Val Lys 180 185 190 Glu Arg Gly Lys Asn Arg Lys Lys Val LeuThr Leu Leu Gly Leu Ser 195 200 205 Ser Leu Ala Ser Trp Val Ser Ile ValHis Leu Trp Ser Asn Gln Leu 210 215 220 Arg Pro Glu Gly Gln Asn His ValIle 225 230 11 489 DNA Homo sapiens 11 atggggcaaa tgaaacatgt ctttgaggtcactttggcat taaagagaca ccagactgga 60 gccaggtggc ggcccctccc acagcgggagagccagggat tgatgggtgg aaatgggaga 120 ggcaccttca cagacagaaa agctcagccaggggacttcc tgggtttgct ggccagaggt 180 accactccca gtcccaccac agctgccccctcctccagat gctggttccg tgaagggaca 240 accatgtacg ccctctatat caccgtccacggctacttcc tcatcacctt cctctttggc 300 atggtggtcc tggccctggt ggtctggaagatcttcaccc tgtcccgtgc tacagcggtc 360 aaggagcggg ggaagaaccg gaagaaggtgctcaccctgc tgggcctctc gagccttgca 420 agttgggtgt ccatcgtcca tctctggtccaatcagctgc gaccagaagg gcagaatcat 480 gtgatatga 489 12 162 PRT Homosapiens 12 Met Gly Gln Met Lys His Val Phe Glu Val Thr Leu Ala Leu LysArg 1 5 10 15 His Gln Thr Gly Ala Arg Trp Arg Pro Leu Pro Gln Arg GluSer Gln 20 25 30 Gly Leu Met Gly Gly Asn Gly Arg Gly Thr Phe Thr Asp ArgLys Ala 35 40 45 Gln Pro Gly Asp Phe Leu Gly Leu Leu Ala Arg Gly Thr ThrPro Ser 50 55 60 Pro Thr Thr Ala Ala Pro Ser Ser Arg Cys Trp Phe Arg GluGly Thr 65 70 75 80 Thr Met Tyr Ala Leu Tyr Ile Thr Val His Gly Tyr PheLeu Ile Thr 85 90 95 Phe Leu Phe Gly Met Val Val Leu Ala Leu Val Val TrpLys Ile Phe 100 105 110 Thr Leu Ser Arg Ala Thr Ala Val Lys Glu Arg GlyLys Asn Arg Lys 115 120 125 Lys Val Leu Thr Leu Leu Gly Leu Ser Ser LeuAla Ser Trp Val Ser 130 135 140 Ile Val His Leu Trp Ser Asn Gln Leu ArgPro Glu Gly Gln Asn His 145 150 155 160 Val Ile 13 1515 DNA Homo sapiens13 atggcgacgc ccaggggcct gggggccctg ctcctgctcc tcctgctccc gacctcaggt 60caggaaaagc ccaccgaagg gccaagaaac acctgcctgg ggagcaacaa catgtacgac 120atcttcaact tgaatgacaa ggctttgtgc ttcaccaagt gcaggcagtc gggcagcgac 180tcctgcaatg tggaaaactt gcagagatac tggctaaact acgaggccca tctgatgaag 240gaaggtttga cgcagaaggt gaacacgcct ttcctgaagg ctttggtcca gaacctcagc 300accaacactg cagaagactt ctatttctct ctggagccct ctcaggttcc gaggcaggtg 360atgaaggacg aggacaagcc ccctgacaga gtgcgacttc ccaagagcct ttttcgatcc 420ctgccaggca acaggtctgt ggtccgcttg gccgtcacca ttctggacat tggtccaggg 480actctcttca agggcccccg gctcggcctg ggagatggca gcggcgtgtt gaacaatcgc 540ctggtgggtt tgagtgtggg acaaatgcat gtcaccaagc tggctgagcc tctggagatc 600gtcttctctc accagcgacc gccccctaac atgaccctca cctgtgtatt ctgggatgtg 660actaaaggga ccactggaga ctggtcttct gagggctgct ccacggaggt cagacctgag 720gggaccgtgt gctgctgtga ccacctgacc tttttcgccc tgctcctgag acccaccttg 780gaccagtcca cggtgcatat cctcacacgc atctcccagg cgggctgtgg ggtctccatg 840atcttcctgg ccttcaccat tattctttat gcctttctga ggctttcccg ggagaggttc 900aagtcagaag atgccccaaa gatccacgtg gccctgggtg gcagcctgtt cctcctgaat 960ctggccttct tggtcaatgt ggggagtggc tcaaaggggt ctgatgctgc ctgctgggcc 1020cggggggctg tcttccacta cttcctgctc tgtgccttca cctggatggg ccttgaagcc 1080ttccacctct acctgctcgc tgtcagggtc ttcaacacct acttcgggca ctacttcctg 1140aagctgagcc tggtgggctg gggcctgccc gccctgatgg tcatcggcac tgggagtgcc 1200aacagctacg gcctctacac catccgtgat agggagaacc gcacctctct ggagctatgc 1260tggttccgtg aagggacaac catgtacgcc ctctatatca ccgtccacgg ctacttcctc 1320atcaccttcc tctttggcat ggtggtcctg gccctggtgg tctggaagat cttcaccctg 1380tcccgtgcta cagcggtcaa ggagcggggg aagaaccggt gctcaccctg ctgggcctct 1440cgagccttgc aagttgggtg tccatcgtcc atctctggtc caatcagctg cgaccagaag 1500ggcagaatca tgtga 1515 14 504 PRT Homo sapiens 14 Met Ala Thr Pro Arg GlyLeu Gly Ala Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Pro Thr Ser Gly GlnGlu Lys Pro Thr Glu Gly Pro Arg Asn Thr Cys 20 25 30 Leu Gly Ser Asn AsnMet Tyr Asp Ile Phe Asn Leu Asn Asp Lys Ala 35 40 45 Leu Cys Phe Thr LysCys Arg Gln Ser Gly Ser Asp Ser Cys Asn Val 50 55 60 Glu Asn Leu Gln ArgTyr Trp Leu Asn Tyr Glu Ala His Leu Met Lys 65 70 75 80 Glu Gly Leu ThrGln Lys Val Asn Thr Pro Phe Leu Lys Ala Leu Val 85 90 95 Gln Asn Leu SerThr Asn Thr Ala Glu Asp Phe Tyr Phe Ser Leu Glu 100 105 110 Pro Ser GlnVal Pro Arg Gln Val Met Lys Asp Glu Asp Lys Pro Pro 115 120 125 Asp ArgVal Arg Leu Pro Lys Ser Leu Phe Arg Ser Leu Pro Gly Asn 130 135 140 ArgSer Val Val Arg Leu Ala Val Thr Ile Leu Asp Ile Gly Pro Gly 145 150 155160 Thr Leu Phe Lys Gly Pro Arg Leu Gly Leu Gly Asp Gly Ser Gly Val 165170 175 Leu Asn Asn Arg Leu Val Gly Leu Ser Val Gly Gln Met His Val Thr180 185 190 Lys Leu Ala Glu Pro Leu Glu Ile Val Phe Ser His Gln Arg ProPro 195 200 205 Pro Asn Met Thr Leu Thr Cys Val Phe Trp Asp Val Thr LysGly Thr 210 215 220 Thr Gly Asp Trp Ser Ser Glu Gly Cys Ser Thr Glu ValArg Pro Glu 225 230 235 240 Gly Thr Val Cys Cys Cys Asp His Leu Thr PhePhe Ala Leu Leu Leu 245 250 255 Arg Pro Thr Leu Asp Gln Ser Thr Val HisIle Leu Thr Arg Ile Ser 260 265 270 Gln Ala Gly Cys Gly Val Ser Met IlePhe Leu Ala Phe Thr Ile Ile 275 280 285 Leu Tyr Ala Phe Leu Arg Leu SerArg Glu Arg Phe Lys Ser Glu Asp 290 295 300 Ala Pro Lys Ile His Val AlaLeu Gly Gly Ser Leu Phe Leu Leu Asn 305 310 315 320 Leu Ala Phe Leu ValAsn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala 325 330 335 Ala Cys Trp AlaArg Gly Ala Val Phe His Tyr Phe Leu Leu Cys Ala 340 345 350 Phe Thr TrpMet Gly Leu Glu Ala Phe His Leu Tyr Leu Leu Ala Val 355 360 365 Arg ValPhe Asn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu 370 375 380 ValGly Trp Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala 385 390 395400 Asn Ser Tyr Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser 405410 415 Leu Glu Leu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr420 425 430 Ile Thr Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly MetVal 435 440 445 Val Leu Ala Leu Val Val Trp Lys Ile Phe Thr Leu Ser ArgAla Thr 450 455 460 Ala Val Lys Glu Arg Gly Lys Asn Arg Cys Ser Pro CysTrp Ala Ser 465 470 475 480 Arg Ala Leu Gln Val Gly Cys Pro Ser Ser IleSer Gly Pro Ile Ser 485 490 495 Cys Asp Gln Lys Gly Arg Ile Met 500 15885 DNA Homo sapiens 15 atgaccctca cctgtgtatt ctgggatgtg actaaagggaccactggaga ctggtcttct 60 gagggctgct ccacggaggt cagacctgag gggaccgtgtgctgctgtga ccacctgacc 120 tttttcgccc tgctcctgag acccaccttg gaccagtccacggtgcatat cctcacacgc 180 atctcccagg cgggctgtgg ggtctccatg atcttcctggccttcaccat tattctttat 240 gcctttctga ggctttcccg ggagaggttc aagtcagaagatgccccaaa gatccacgtg 300 gccctgggtg gcagcctgtt cctcctgaat ctggccttcttggtcaatgt ggggagtggc 360 tcaaaggggt ctgatgctgc ctgctgggcc cggggggctgtcttccacta cttcctgctc 420 tgtgccttca cctggatggg ccttgaagcc ttccacctctacctgctcgc tgtcagggtc 480 ttcaacacct acttcgggca ctacttcctg aagctgagcctggtgggctg gggcctgccc 540 gccctgatgg tcatcggcac tgggagtgcc aacagctacggcctctacac catccgtgat 600 agggagaacc gcacctctct ggagctatgc tggttccgtgaagggacaac catgtacgcc 660 ctctatatca ccgtccacgg ctacttcctc atcaccttcctctttggcat ggtggtcctg 720 gccctggtgg tctggaagat cttcaccctg tcccgtgctacagcggtcaa ggagcggggg 780 aagaaccggt gctcaccctg ctgggcctct cgagccttgcaagttgggtg tccatcgtcc 840 atctctggtc caatcagctg cgaccagaag ggcagaatcatgtga 885 16 294 PRT Homo sapiens 16 Met Thr Leu Thr Cys Val Phe Trp AspVal Thr Lys Gly Thr Thr Gly 1 5 10 15 Asp Trp Ser Ser Glu Gly Cys SerThr Glu Val Arg Pro Glu Gly Thr 20 25 30 Val Cys Cys Cys Asp His Leu ThrPhe Phe Ala Leu Leu Leu Arg Pro 35 40 45 Thr Leu Asp Gln Ser Thr Val HisIle Leu Thr Arg Ile Ser Gln Ala 50 55 60 Gly Cys Gly Val Ser Met Ile PheLeu Ala Phe Thr Ile Ile Leu Tyr 65 70 75 80 Ala Phe Leu Arg Leu Ser ArgGlu Arg Phe Lys Ser Glu Asp Ala Pro 85 90 95 Lys Ile His Val Ala Leu GlyGly Ser Leu Phe Leu Leu Asn Leu Ala 100 105 110 Phe Leu Val Asn Val GlySer Gly Ser Lys Gly Ser Asp Ala Ala Cys 115 120 125 Trp Ala Arg Gly AlaVal Phe His Tyr Phe Leu Leu Cys Ala Phe Thr 130 135 140 Trp Met Gly LeuGlu Ala Phe His Leu Tyr Leu Leu Ala Val Arg Val 145 150 155 160 Phe AsnThr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu Val Gly 165 170 175 TrpGly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser 180 185 190Tyr Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu 195 200205 Leu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr 210215 220 Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val Val Leu225 230 235 240 Ala Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg Ala ThrAla Val 245 250 255 Lys Glu Arg Gly Lys Asn Arg Cys Ser Pro Cys Trp AlaSer Arg Ala 260 265 270 Leu Gln Val Gly Cys Pro Ser Ser Ile Ser Gly ProIle Ser Cys Asp 275 280 285 Gln Lys Gly Arg Ile Met 290 17 1068 DNA Homosapiens 17 atggcccctt ctgcagcctg gcctccccga tctccccttt cacagggcccccggctcggc 60 ctgggagatg gcagcggcgt gttgaacaat cgcctggtgg gtttgagtgtgggacaaatg 120 catgtcacca agctggctga gcctctggag atcgtcttct ctcaccagcgaccgccccct 180 aacatgaccc tcacctgtgt attctgggat gtgactaaag ggaccactggagactggtct 240 tctgagggct gctccacgga ggtcagacct gaggggaccg tgtgctgctgtgaccacctg 300 acctttttcg ccctgctcct gagacccacc ttggaccagt ccacggtgcatatcctcaca 360 cgcatctccc aggcgggctg tggggtctcc atgatcttcc tggccttcaccattattctt 420 tatgcctttc tgaggctttc ccgggagagg ttcaagtcag aagatgccccaaagatccac 480 gtggccctgg gtggcagcct gttcctcctg aatctggcct tcttggtcaatgtggggagt 540 ggctcaaagg ggtctgatgc tgcctgctgg gcccgggggg ctgtcttccactacttcctg 600 ctctgtgcct tcacctggat gggccttgaa gccttccacc tctacctgctcgctgtcagg 660 gtcttcaaca cctacttcgg gcactacttc ctgaagctga gcctggtgggctggggcctg 720 cccgccctga tggtcatcgg cactgggagt gccaacagct acggcctctacaccatccgt 780 gatagggaga accgcacctc tctggagcta tgctggttcc gtgaagggacaaccatgtac 840 gccctctata tcaccgtcca cggctacttc ctcatcacct tcctctttggcatggtggtc 900 ctggccctgg tggtctggaa gatcttcacc ctgtcccgtg ctacagcggtcaaggagcgg 960 gggaagaacc ggtgctcacc ctgctgggcc tctcgagcct tgcaagttgggtgtccatcg 1020 tccatctctg gtccaatcag ctgcgaccag aagggcagaa tcatgtga1068 18 355 PRT Homo sapiens 18 Met Ala Pro Ser Ala Ala Trp Pro Pro ArgSer Pro Leu Ser Gln Gly 1 5 10 15 Pro Arg Leu Gly Leu Gly Asp Gly SerGly Val Leu Asn Asn Arg Leu 20 25 30 Val Gly Leu Ser Val Gly Gln Met HisVal Thr Lys Leu Ala Glu Pro 35 40 45 Leu Glu Ile Val Phe Ser His Gln ArgPro Pro Pro Asn Met Thr Leu 50 55 60 Thr Cys Val Phe Trp Asp Val Thr LysGly Thr Thr Gly Asp Trp Ser 65 70 75 80 Ser Glu Gly Cys Ser Thr Glu ValArg Pro Glu Gly Thr Val Cys Cys 85 90 95 Cys Asp His Leu Thr Phe Phe AlaLeu Leu Leu Arg Pro Thr Leu Asp 100 105 110 Gln Ser Thr Val His Ile LeuThr Arg Ile Ser Gln Ala Gly Cys Gly 115 120 125 Val Ser Met Ile Phe LeuAla Phe Thr Ile Ile Leu Tyr Ala Phe Leu 130 135 140 Arg Leu Ser Arg GluArg Phe Lys Ser Glu Asp Ala Pro Lys Ile His 145 150 155 160 Val Ala LeuGly Gly Ser Leu Phe Leu Leu Asn Leu Ala Phe Leu Val 165 170 175 Asn ValGly Ser Gly Ser Lys Gly Ser Asp Ala Ala Cys Trp Ala Arg 180 185 190 GlyAla Val Phe His Tyr Phe Leu Leu Cys Ala Phe Thr Trp Met Gly 195 200 205Leu Glu Ala Phe His Leu Tyr Leu Leu Ala Val Arg Val Phe Asn Thr 210 215220 Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu Val Gly Trp Gly Leu 225230 235 240 Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser Tyr GlyLeu 245 250 255 Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu LeuCys Trp 260 265 270 Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile ThrVal His Gly 275 280 285 Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val ValLeu Ala Leu Val 290 295 300 Val Trp Lys Ile Phe Thr Leu Ser Arg Ala ThrAla Val Lys Glu Arg 305 310 315 320 Gly Lys Asn Arg Cys Ser Pro Cys TrpAla Ser Arg Ala Leu Gln Val 325 330 335 Gly Cys Pro Ser Ser Ile Ser GlyPro Ile Ser Cys Asp Gln Lys Gly 340 345 350 Arg Ile Met 355 19 690 DNAHomo sapiens 19 atgggagctc cccatgggag ctgtggcccc ttggggcctc ttatttctcaccccaggctt 60 tcccgggaga ggttcaagtc agaagatgcc ccaaagatcc acgtggccctgggtggcagc 120 ctgttcctcc tgaatctggc cttcttggtc aatgtgggga gtggctcaaaggggtctgat 180 gctgcctgct gggcccgggg ggctgtcttc cactacttcc tgctctgtgccttcacctgg 240 atgggccttg aagccttcca cctctacctg ctcgctgtca gggtcttcaacacctacttc 300 gggcactact tcctgaagct gagcctggtg ggctggggcc tgcccgccctgatggtcatc 360 ggcactggga gtgccaacag ctacggcctc tacaccatcc gtgatagggagaaccgcacc 420 tctctggagc tatgctggtt ccgtgaaggg acaaccatgt acgccctctatatcaccgtc 480 cacggctact tcctcatcac cttcctcttt ggcatggtgg tcctggccctggtggtctgg 540 aagatcttca ccctgtcccg tgctacagcg gtcaaggagc gggggaagaaccggtgctca 600 ccctgctggg cctctcgagc cttgcaagtt gggtgtccat cgtccatctctggtccaatc 660 agctgcgacc agaagggcag aatcatgtga 690 20 229 PRT Homosapiens 20 Met Gly Ala Pro His Gly Ser Cys Gly Pro Leu Gly Pro Leu IleSer 1 5 10 15 His Pro Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp AlaPro Lys 20 25 30 Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn LeuAla Phe 35 40 45 Leu Val Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala AlaCys Trp 50 55 60 Ala Arg Gly Ala Val Phe His Tyr Phe Leu Leu Cys Ala PheThr Trp 65 70 75 80 Met Gly Leu Glu Ala Phe His Leu Tyr Leu Leu Ala ValArg Val Phe 85 90 95 Asn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser LeuVal Gly Trp 100 105 110 Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly SerAla Asn Ser Tyr 115 120 125 Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn ArgThr Ser Leu Glu Leu 130 135 140 Cys Trp Phe Arg Glu Gly Thr Thr Met TyrAla Leu Tyr Ile Thr Val 145 150 155 160 His Gly Tyr Phe Leu Ile Thr PheLeu Phe Gly Met Val Val Leu Ala 165 170 175 Leu Val Val Trp Lys Ile PheThr Leu Ser Arg Ala Thr Ala Val Lys 180 185 190 Glu Arg Gly Lys Asn ArgCys Ser Pro Cys Trp Ala Ser Arg Ala Leu 195 200 205 Gln Val Gly Cys ProSer Ser Ile Ser Gly Pro Ile Ser Cys Asp Gln 210 215 220 Lys Gly Arg IleMet 225 21 477 DNA Homo sapiens 21 atggggcaaa tgaaacatgt ctttgaggtcactttggcat taaagagaca ccagactgga 60 gccaggtggc ggcccctccc acagcgggagagccagggat tgatgggtgg aaatgggaga 120 ggcaccttca cagacagaaa agctcagccaggggacttcc tgggtttgct ggccagaggt 180 accactccca gtcccaccac agctgccccctcctccagat gctggttccg tgaagggaca 240 accatgtacg ccctctatat caccgtccacggctacttcc tcatcacctt cctctttggc 300 atggtggtcc tggccctggt ggtctggaagatcttcaccc tgtcccgtgc tacagcggtc 360 aaggagcggg ggaagaaccg gtgctcaccctgctgggcct ctcgagcctt gcaagttggg 420 tgtccatcgt ccatctctgg tccaatcagctgcgaccaga agggcagaat catgtga 477 22 158 PRT Homo sapiens 22 Met Gly GlnMet Lys His Val Phe Glu Val Thr Leu Ala Leu Lys Arg 1 5 10 15 His GlnThr Gly Ala Arg Trp Arg Pro Leu Pro Gln Arg Glu Ser Gln 20 25 30 Gly LeuMet Gly Gly Asn Gly Arg Gly Thr Phe Thr Asp Arg Lys Ala 35 40 45 Gln ProGly Asp Phe Leu Gly Leu Leu Ala Arg Gly Thr Thr Pro Ser 50 55 60 Pro ThrThr Ala Ala Pro Ser Ser Arg Cys Trp Phe Arg Glu Gly Thr 65 70 75 80 ThrMet Tyr Ala Leu Tyr Ile Thr Val His Gly Tyr Phe Leu Ile Thr 85 90 95 PheLeu Phe Gly Met Val Val Leu Ala Leu Val Val Trp Lys Ile Phe 100 105 110Thr Leu Ser Arg Ala Thr Ala Val Lys Glu Arg Gly Lys Asn Arg Cys 115 120125 Ser Pro Cys Trp Ala Ser Arg Ala Leu Gln Val Gly Cys Pro Ser Ser 130135 140 Ile Ser Gly Pro Ile Ser Cys Asp Gln Lys Gly Arg Ile Met 145 150155 23 1566 DNA Homo sapiens 23 atggcgacgc ccaggggcct gggggccctgctcctgctcc tcctgctccc gacctcaggt 60 caggaaaagc ccaccgaagg gccaagaaacacctgcctgg ggagcaacaa catgtacgac 120 atcttcaact tgaatgacaa ggctttgtgcttcaccaagt gcaggcagtc gggcagcgac 180 tcctgcaatg tggaaaactt gcagagatactggctaaact acgaggccca tctgatgaag 240 gaaggtttga cgcagaaggt gaacacgcctttcctgaagg ctttggtcca gaacctcagc 300 accaacactg cagaagactt ctatttctctctggagccct ctcaggttcc gaggcaggtg 360 atgaaggacg aggacaagcc ccctgacagagtgcgacttc ccaagagcct ttttcgatcc 420 ctgccaggca acaggtctgt ggtccgcttggccgtcacca ttctggacat tggtccaggg 480 actctcttca agggcccccg gctcggcctgggagatggca gcggcgtgtt gaacaatcgc 540 ctggtgggtt tgagtgtggg acaaatgcatgtcaccaagc tggctgagcc tctggagatc 600 gtcttctctc accagcgacc gccccctaacatgaccctca cctgtgtatt ctgggatgtg 660 actaaaggga ccactggaga ctggtcttctgagggctgct ccacggaggt cagacctgag 720 gggaccgtgt gctgctgtga ccacctgacctttttcgccc tgctcctgag acccaccttg 780 gaccagtcca cggtgcatat cctcacacgcatctcccagg cgggctgtgg ggtctccatg 840 atcttcctgg ccttcaccat tattctttatgcctttctga ggctttcccg ggagaggttc 900 aagtcagaag atgccccaaa gatccacgtggccctgggtg gcagcctgtt cctcctgaat 960 ctggccttct tggtcaatgt ggggagtggctcaaaggggt ctgatgctgc ctgctgggcc 1020 cggggggctg tcttccacta cttcctgctctgtgccttca cctggatggg ccttgaagcc 1080 ttccacctct acctgctcgc tgtcagggtcttcaacacct acttcgggca ctacttcctg 1140 aagctgagcc tggtgggctg gggcctgcccgccctgatgg tcatcggcac tgggagtgcc 1200 aacagctacg gcctctacac catccgtgatagggagaacc gcacctctct ggagctatgc 1260 tggttccgtg aagggacaac catgtacgccctctatatca ccgtccacgg ctacttcctc 1320 atcaccttcc tctttggcat ggtggtcctggccctggtgg tctggaagat cttcaccctg 1380 tcccgtgcta cagcggtcaa ggagcgggggaagaaccgga agaaggtgct caccctgctg 1440 ggcctctcga gcctggtggg tgtgacatgggggttggcca tcttcacccc gttgggcctc 1500 tccaccgtct acatctttgc acttttcaactccttgcaag gtgaggcccc tgcaccaggg 1560 aggtga 1566 24 521 PRT Homosapiens 24 Met Ala Thr Pro Arg Gly Leu Gly Ala Leu Leu Leu Leu Leu LeuLeu 1 5 10 15 Pro Thr Ser Gly Gln Glu Lys Pro Thr Glu Gly Pro Arg AsnThr Cys 20 25 30 Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu Asn AspLys Ala 35 40 45 Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp Ser CysAsn Val 50 55 60 Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala His LeuMet Lys 65 70 75 80 Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe Leu LysAla Leu Val 85 90 95 Gln Asn Leu Ser Thr Asn Thr Ala Glu Asp Phe Tyr PheSer Leu Glu 100 105 110 Pro Ser Gln Val Pro Arg Gln Val Met Lys Asp GluAsp Lys Pro Pro 115 120 125 Asp Arg Val Arg Leu Pro Lys Ser Leu Phe ArgSer Leu Pro Gly Asn 130 135 140 Arg Ser Val Val Arg Leu Ala Val Thr IleLeu Asp Ile Gly Pro Gly 145 150 155 160 Thr Leu Phe Lys Gly Pro Arg LeuGly Leu Gly Asp Gly Ser Gly Val 165 170 175 Leu Asn Asn Arg Leu Val GlyLeu Ser Val Gly Gln Met His Val Thr 180 185 190 Lys Leu Ala Glu Pro LeuGlu Ile Val Phe Ser His Gln Arg Pro Pro 195 200 205 Pro Asn Met Thr LeuThr Cys Val Phe Trp Asp Val Thr Lys Gly Thr 210 215 220 Thr Gly Asp TrpSer Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu 225 230 235 240 Gly ThrVal Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu 245 250 255 ArgPro Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser 260 265 270Gln Ala Gly Cys Gly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile 275 280285 Leu Tyr Ala Phe Leu Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp 290295 300 Ala Pro Lys Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn305 310 315 320 Leu Ala Phe Leu Val Asn Val Gly Ser Gly Ser Lys Gly SerAsp Ala 325 330 335 Ala Cys Trp Ala Arg Gly Ala Val Phe His Tyr Phe LeuLeu Cys Ala 340 345 350 Phe Thr Trp Met Gly Leu Glu Ala Phe His Leu TyrLeu Leu Ala Val 355 360 365 Arg Val Phe Asn Thr Tyr Phe Gly His Tyr PheLeu Lys Leu Ser Leu 370 375 380 Val Gly Trp Gly Leu Pro Ala Leu Met ValIle Gly Thr Gly Ser Ala 385 390 395 400 Asn Ser Tyr Gly Leu Tyr Thr IleArg Asp Arg Glu Asn Arg Thr Ser 405 410 415 Leu Glu Leu Cys Trp Phe ArgGlu Gly Thr Thr Met Tyr Ala Leu Tyr 420 425 430 Ile Thr Val His Gly TyrPhe Leu Ile Thr Phe Leu Phe Gly Met Val 435 440 445 Val Leu Ala Leu ValVal Trp Lys Ile Phe Thr Leu Ser Arg Ala Thr 450 455 460 Ala Val Lys GluArg Gly Lys Asn Arg Lys Lys Val Leu Thr Leu Leu 465 470 475 480 Gly LeuSer Ser Leu Val Gly Val Thr Trp Gly Leu Ala Ile Phe Thr 485 490 495 ProLeu Gly Leu Ser Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu 500 505 510Gln Gly Glu Ala Pro Ala Pro Gly Arg 515 520 25 936 DNA Homo sapiens 25atgaccctca cctgtgtatt ctgggatgtg actaaaggga ccactggaga ctggtcttct 60gagggctgct ccacggaggt cagacctgag gggaccgtgt gctgctgtga ccacctgacc 120tttttcgccc tgctcctgag acccaccttg gaccagtcca cggtgcatat cctcacacgc 180atctcccagg cgggctgtgg ggtctccatg atcttcctgg ccttcaccat tattctttat 240gcctttctga ggctttcccg ggagaggttc aagtcagaag atgccccaaa gatccacgtg 300gccctgggtg gcagcctgtt cctcctgaat ctggccttct tggtcaatgt ggggagtggc 360tcaaaggggt ctgatgctgc ctgctgggcc cggggggctg tcttccacta cttcctgctc 420tgtgccttca cctggatggg ccttgaagcc ttccacctct acctgctcgc tgtcagggtc 480ttcaacacct acttcgggca ctacttcctg aagctgagcc tggtgggctg gggcctgccc 540gccctgatgg tcatcggcac tgggagtgcc aacagctacg gcctctacac catccgtgat 600agggagaacc gcacctctct ggagctatgc tggttccgtg aagggacaac catgtacgcc 660ctctatatca ccgtccacgg ctacttcctc atcaccttcc tctttggcat ggtggtcctg 720gccctggtgg tctggaagat cttcaccctg tcccgtgcta cagcggtcaa ggagcggggg 780aagaaccgga agaaggtgct caccctgctg ggcctctcga gcctggtggg tgtgacatgg 840gggttggcca tcttcacccc gttgggcctc tccaccgtct acatctttgc acttttcaac 900tccttgcaag gtgaggcccc tgcaccaggg aggtga 936 26 311 PRT Homo sapiens 26Met Thr Leu Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr Thr Gly 1 5 1015 Asp Trp Ser Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu Gly Thr 20 2530 Val Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu Arg Pro 35 4045 Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser Gln Ala 50 5560 Gly Cys Gly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile Leu Tyr 65 7075 80 Ala Phe Leu Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp Ala Pro 8590 95 Lys Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn Leu Ala100 105 110 Phe Leu Val Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala AlaCys 115 120 125 Trp Ala Arg Gly Ala Val Phe His Tyr Phe Leu Leu Cys AlaPhe Thr 130 135 140 Trp Met Gly Leu Glu Ala Phe His Leu Tyr Leu Leu AlaVal Arg Val 145 150 155 160 Phe Asn Thr Tyr Phe Gly His Tyr Phe Leu LysLeu Ser Leu Val Gly 165 170 175 Trp Gly Leu Pro Ala Leu Met Val Ile GlyThr Gly Ser Ala Asn Ser 180 185 190 Tyr Gly Leu Tyr Thr Ile Arg Asp ArgGlu Asn Arg Thr Ser Leu Glu 195 200 205 Leu Cys Trp Phe Arg Glu Gly ThrThr Met Tyr Ala Leu Tyr Ile Thr 210 215 220 Val His Gly Tyr Phe Leu IleThr Phe Leu Phe Gly Met Val Val Leu 225 230 235 240 Ala Leu Val Val TrpLys Ile Phe Thr Leu Ser Arg Ala Thr Ala Val 245 250 255 Lys Glu Arg GlyLys Asn Arg Lys Lys Val Leu Thr Leu Leu Gly Leu 260 265 270 Ser Ser LeuVal Gly Val Thr Trp Gly Leu Ala Ile Phe Thr Pro Leu 275 280 285 Gly LeuSer Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu Gln Gly 290 295 300 GluAla Pro Ala Pro Gly Arg 305 310 27 1119 DNA Homo sapiens 27 atggccccttctgcagcctg gcctccccga tctccccttt cacagggccc ccggctcggc 60 ctgggagatggcagcggcgt gttgaacaat cgcctggtgg gtttgagtgt gggacaaatg 120 catgtcaccaagctggctga gcctctggag atcgtcttct ctcaccagcg accgccccct 180 aacatgaccctcacctgtgt attctgggat gtgactaaag ggaccactgg agactggtct 240 tctgagggctgctccacgga ggtcagacct gaggggaccg tgtgctgctg tgaccacctg 300 acctttttcgccctgctcct gagacccacc ttggaccagt ccacggtgca tatcctcaca 360 cgcatctcccaggcgggctg tggggtctcc atgatcttcc tggccttcac cattattctt 420 tatgcctttctgaggctttc ccgggagagg ttcaagtcag aagatgcccc aaagatccac 480 gtggccctgggtggcagcct gttcctcctg aatctggcct tcttggtcaa tgtggggagt 540 ggctcaaaggggtctgatgc tgcctgctgg gcccgggggg ctgtcttcca ctacttcctg 600 ctctgtgccttcacctggat gggccttgaa gccttccacc tctacctgct cgctgtcagg 660 gtcttcaacacctacttcgg gcactacttc ctgaagctga gcctggtggg ctggggcctg 720 cccgccctgatggtcatcgg cactgggagt gccaacagct acggcctcta caccatccgt 780 gatagggagaaccgcacctc tctggagcta tgctggttcc gtgaagggac aaccatgtac 840 gccctctatatcaccgtcca cggctacttc ctcatcacct tcctctttgg catggtggtc 900 ctggccctggtggtctggaa gatcttcacc ctgtcccgtg ctacagcggt caaggagcgg 960 gggaagaaccggaagaaggt gctcaccctg ctgggcctct cgagcctggt gggtgtgaca 1020 tgggggttggccatcttcac cccgttgggc ctctccaccg tctacatctt tgcacttttc 1080 aactccttgcaaggtgaggc ccctgcacca gggaggtga 1119 28 372 PRT Homo sapiens 28 Met AlaPro Ser Ala Ala Trp Pro Pro Arg Ser Pro Leu Ser Gln Gly 1 5 10 15 ProArg Leu Gly Leu Gly Asp Gly Ser Gly Val Leu Asn Asn Arg Leu 20 25 30 ValGly Leu Ser Val Gly Gln Met His Val Thr Lys Leu Ala Glu Pro 35 40 45 LeuGlu Ile Val Phe Ser His Gln Arg Pro Pro Pro Asn Met Thr Leu 50 55 60 ThrCys Val Phe Trp Asp Val Thr Lys Gly Thr Thr Gly Asp Trp Ser 65 70 75 80Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu Gly Thr Val Cys Cys 85 90 95Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu Arg Pro Thr Leu Asp 100 105110 Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser Gln Ala Gly Cys Gly 115120 125 Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile Leu Tyr Ala Phe Leu130 135 140 Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp Ala Pro Lys IleHis 145 150 155 160 Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn Leu AlaPhe Leu Val 165 170 175 Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala AlaCys Trp Ala Arg 180 185 190 Gly Ala Val Phe His Tyr Phe Leu Leu Cys AlaPhe Thr Trp Met Gly 195 200 205 Leu Glu Ala Phe His Leu Tyr Leu Leu AlaVal Arg Val Phe Asn Thr 210 215 220 Tyr Phe Gly His Tyr Phe Leu Lys LeuSer Leu Val Gly Trp Gly Leu 225 230 235 240 Pro Ala Leu Met Val Ile GlyThr Gly Ser Ala Asn Ser Tyr Gly Leu 245 250 255 Tyr Thr Ile Arg Asp ArgGlu Asn Arg Thr Ser Leu Glu Leu Cys Trp 260 265 270 Phe Arg Glu Gly ThrThr Met Tyr Ala Leu Tyr Ile Thr Val His Gly 275 280 285 Tyr Phe Leu IleThr Phe Leu Phe Gly Met Val Val Leu Ala Leu Val 290 295 300 Val Trp LysIle Phe Thr Leu Ser Arg Ala Thr Ala Val Lys Glu Arg 305 310 315 320 GlyLys Asn Arg Lys Lys Val Leu Thr Leu Leu Gly Leu Ser Ser Leu 325 330 335Val Gly Val Thr Trp Gly Leu Ala Ile Phe Thr Pro Leu Gly Leu Ser 340 345350 Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu Gln Gly Glu Ala Pro 355360 365 Ala Pro Gly Arg 370 29 741 DNA Homo sapiens 29 atgggagctccccatgggag ctgtggcccc ttggggcctc ttatttctca ccccaggctt 60 tcccgggagaggttcaagtc agaagatgcc ccaaagatcc acgtggccct gggtggcagc 120 ctgttcctcctgaatctggc cttcttggtc aatgtgggga gtggctcaaa ggggtctgat 180 gctgcctgctgggcccgggg ggctgtcttc cactacttcc tgctctgtgc cttcacctgg 240 atgggccttgaagccttcca cctctacctg ctcgctgtca gggtcttcaa cacctacttc 300 gggcactacttcctgaagct gagcctggtg ggctggggcc tgcccgccct gatggtcatc 360 ggcactgggagtgccaacag ctacggcctc tacaccatcc gtgataggga gaaccgcacc 420 tctctggagctatgctggtt ccgtgaaggg acaaccatgt acgccctcta tatcaccgtc 480 cacggctacttcctcatcac cttcctcttt ggcatggtgg tcctggccct ggtggtctgg 540 aagatcttcaccctgtcccg tgctacagcg gtcaaggagc gggggaagaa ccggaagaag 600 gtgctcaccctgctgggcct ctcgagcctg gtgggtgtga catgggggtt ggccatcttc 660 accccgttgggcctctccac cgtctacatc tttgcacttt tcaactcctt gcaaggtgag 720 gcccctgcaccagggaggtg a 741 30 246 PRT Homo sapiens 30 Met Gly Ala Pro His Gly SerCys Gly Pro Leu Gly Pro Leu Ile Ser 1 5 10 15 His Pro Arg Leu Ser ArgGlu Arg Phe Lys Ser Glu Asp Ala Pro Lys 20 25 30 Ile His Val Ala Leu GlyGly Ser Leu Phe Leu Leu Asn Leu Ala Phe 35 40 45 Leu Val Asn Val Gly SerGly Ser Lys Gly Ser Asp Ala Ala Cys Trp 50 55 60 Ala Arg Gly Ala Val PheHis Tyr Phe Leu Leu Cys Ala Phe Thr Trp 65 70 75 80 Met Gly Leu Glu AlaPhe His Leu Tyr Leu Leu Ala Val Arg Val Phe 85 90 95 Asn Thr Tyr Phe GlyHis Tyr Phe Leu Lys Leu Ser Leu Val Gly Trp 100 105 110 Gly Leu Pro AlaLeu Met Val Ile Gly Thr Gly Ser Ala Asn Ser Tyr 115 120 125 Gly Leu TyrThr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu Leu 130 135 140 Cys TrpPhe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr Val 145 150 155 160His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val Val Leu Ala 165 170175 Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg Ala Thr Ala Val Lys 180185 190 Glu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr Leu Leu Gly Leu Ser195 200 205 Ser Leu Val Gly Val Thr Trp Gly Leu Ala Ile Phe Thr Pro LeuGly 210 215 220 Leu Ser Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu GlnGly Glu 225 230 235 240 Ala Pro Ala Pro Gly Arg 245 31 528 DNA Homosapiens 31 atggggcaaa tgaaacatgt ctttgaggtc actttggcat taaagagacaccagactgga 60 gccaggtggc ggcccctccc acagcgggag agccagggat tgatgggtggaaatgggaga 120 ggcaccttca cagacagaaa agctcagcca ggggacttcc tgggtttgctggccagaggt 180 accactccca gtcccaccac agctgccccc tcctccagat gctggttccgtgaagggaca 240 accatgtacg ccctctatat caccgtccac ggctacttcc tcatcaccttcctctttggc 300 atggtggtcc tggccctggt ggtctggaag atcttcaccc tgtcccgtgctacagcggtc 360 aaggagcggg ggaagaaccg gaagaaggtg ctcaccctgc tgggcctctcgagcctggtg 420 ggtgtgacat gggggttggc catcttcacc ccgttgggcc tctccaccgtctacatcttt 480 gcacttttca actccttgca aggtgaggcc cctgcaccag ggaggtga 52832 175 PRT Homo sapiens 32 Met Gly Gln Met Lys His Val Phe Glu Val ThrLeu Ala Leu Lys Arg 1 5 10 15 His Gln Thr Gly Ala Arg Trp Arg Pro LeuPro Gln Arg Glu Ser Gln 20 25 30 Gly Leu Met Gly Gly Asn Gly Arg Gly ThrPhe Thr Asp Arg Lys Ala 35 40 45 Gln Pro Gly Asp Phe Leu Gly Leu Leu AlaArg Gly Thr Thr Pro Ser 50 55 60 Pro Thr Thr Ala Ala Pro Ser Ser Arg CysTrp Phe Arg Glu Gly Thr 65 70 75 80 Thr Met Tyr Ala Leu Tyr Ile Thr ValHis Gly Tyr Phe Leu Ile Thr 85 90 95 Phe Leu Phe Gly Met Val Val Leu AlaLeu Val Val Trp Lys Ile Phe 100 105 110 Thr Leu Ser Arg Ala Thr Ala ValLys Glu Arg Gly Lys Asn Arg Lys 115 120 125 Lys Val Leu Thr Leu Leu GlyLeu Ser Ser Leu Val Gly Val Thr Trp 130 135 140 Gly Leu Ala Ile Phe ThrPro Leu Gly Leu Ser Thr Val Tyr Ile Phe 145 150 155 160 Ala Leu Phe AsnSer Leu Gln Gly Glu Ala Pro Ala Pro Gly Arg 165 170 175 33 1458 DNA Homosapiens 33 atggcgacgc ccaggggcct gggggccctg ctcctgctcc tcctgctcccgacctcaggt 60 caggaaaagc ccaccgaagg gccaagaaac acctgcctgg ggagcaacaacatgtacgac 120 atcttcaact tgaatgacaa ggctttgtgc ttcaccaagt gcaggcagtcgggcagcgac 180 tcctgcaatg tggaaaactt gcagagatac tggctaaact acgaggcccatctgatgaag 240 gaaggtttga cgcagaaggt gaacacgcct ttcctgaagg ctttggtccagaacctcagc 300 accaacactg cagaagactt ctatttctct ctggagccct ctcaggttccgaggcaggtg 360 atgaaggacg aggacaagcc ccctgacaga gtgcgacttc ccaagagcctttttcgatcc 420 ctgccaggca acaggtctgt ggtccgcttg gccgtcacca ttctggacattggtccaggg 480 actctcttca agggcccccg gctcggcctg ggagatggca gcggcgtgttgaacaatcgc 540 ctggtgggtt tgagtgtggg acaaatgcat gtcaccaagc tggctgagcctctggagatc 600 gtcttctctc accagcgacc gccccctaac atgaccctca cctgtgtattctgggatgtg 660 actaaaggga ccactggaga ctggtcttct gagggctgct ccacggaggtcagacctgag 720 gggaccgtgt gctgctgtga ccacctgacc tttttcgccc tgctcctgagacccaccttg 780 gaccagtcca cggtgcatat cctcacacgc atctcccagg cgggctgtggggtctccatg 840 atcttcctgg ccttcaccat tattctttat gcctttctga ggctttcccgggagaggttc 900 aagtcagaag atgccccaaa gatccacgtg gccctgggtg gcagcctgttcctcctgaat 960 ctggccttct tggtcaatgt ggggagtggc tcaaaggggt ctgatgctgcctgctgggcc 1020 cggggggctg tcttccacta cttcctgctc tgtgccttca cctggatgggccttgaagcc 1080 ttccacctct acctgctcgc tgtcagggtc ttcaacacct acttcgggcactacttcctg 1140 aagctgagcc tggtgggctg gggcctgccc gccctgatgg tcatcggcactgggagtgcc 1200 aacagctacg gcctctacac catccgtgat agggagaacc gcacctctctggagctatgc 1260 tggttccgtg aagggacaac catgtacgcc ctctatatca ccgtccacggctacttcctc 1320 atcaccttcc tctttggcat ggtggtcctg gccctggtgg tctggaagatcttcaccctg 1380 tcccgtgcta cagcggtcaa ggagcggggg aagaaccggt gctcaccctgctgggcctct 1440 cgagcctggt gggtgtga 1458 34 485 PRT Homo sapiens 34 MetAla Thr Pro Arg Gly Leu Gly Ala Leu Leu Leu Leu Leu Leu Leu 1 5 10 15Pro Thr Ser Gly Gln Glu Lys Pro Thr Glu Gly Pro Arg Asn Thr Cys 20 25 30Leu Gly Ser Asn Asn Met Tyr Asp Ile Phe Asn Leu Asn Asp Lys Ala 35 40 45Leu Cys Phe Thr Lys Cys Arg Gln Ser Gly Ser Asp Ser Cys Asn Val 50 55 60Glu Asn Leu Gln Arg Tyr Trp Leu Asn Tyr Glu Ala His Leu Met Lys 65 70 7580 Glu Gly Leu Thr Gln Lys Val Asn Thr Pro Phe Leu Lys Ala Leu Val 85 9095 Gln Asn Leu Ser Thr Asn Thr Ala Glu Asp Phe Tyr Phe Ser Leu Glu 100105 110 Pro Ser Gln Val Pro Arg Gln Val Met Lys Asp Glu Asp Lys Pro Pro115 120 125 Asp Arg Val Arg Leu Pro Lys Ser Leu Phe Arg Ser Leu Pro GlyAsn 130 135 140 Arg Ser Val Val Arg Leu Ala Val Thr Ile Leu Asp Ile GlyPro Gly 145 150 155 160 Thr Leu Phe Lys Gly Pro Arg Leu Gly Leu Gly AspGly Ser Gly Val 165 170 175 Leu Asn Asn Arg Leu Val Gly Leu Ser Val GlyGln Met His Val Thr 180 185 190 Lys Leu Ala Glu Pro Leu Glu Ile Val PheSer His Gln Arg Pro Pro 195 200 205 Pro Asn Met Thr Leu Thr Cys Val PheTrp Asp Val Thr Lys Gly Thr 210 215 220 Thr Gly Asp Trp Ser Ser Glu GlyCys Ser Thr Glu Val Arg Pro Glu 225 230 235 240 Gly Thr Val Cys Cys CysAsp His Leu Thr Phe Phe Ala Leu Leu Leu 245 250 255 Arg Pro Thr Leu AspGln Ser Thr Val His Ile Leu Thr Arg Ile Ser 260 265 270 Gln Ala Gly CysGly Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile 275 280 285 Leu Tyr AlaPhe Leu Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp 290 295 300 Ala ProLys Ile His Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn 305 310 315 320Leu Ala Phe Leu Val Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala 325 330335 Ala Cys Trp Ala Arg Gly Ala Val Phe His Tyr Phe Leu Leu Cys Ala 340345 350 Phe Thr Trp Met Gly Leu Glu Ala Phe His Leu Tyr Leu Leu Ala Val355 360 365 Arg Val Phe Asn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu SerLeu 370 375 380 Val Gly Trp Gly Leu Pro Ala Leu Met Val Ile Gly Thr GlySer Ala 385 390 395 400 Asn Ser Tyr Gly Leu Tyr Thr Ile Arg Asp Arg GluAsn Arg Thr Ser 405 410 415 Leu Glu Leu Cys Trp Phe Arg Glu Gly Thr ThrMet Tyr Ala Leu Tyr 420 425 430 Ile Thr Val His Gly Tyr Phe Leu Ile ThrPhe Leu Phe Gly Met Val 435 440 445 Val Leu Ala Leu Val Val Trp Lys IlePhe Thr Leu Ser Arg Ala Thr 450 455 460 Ala Val Lys Glu Arg Gly Lys AsnArg Cys Ser Pro Cys Trp Ala Ser 465 470 475 480 Arg Ala Trp Trp Val 48535 828 DNA Homo sapiens 35 atgaccctca cctgtgtatt ctgggatgtg actaaagggaccactggaga ctggtcttct 60 gagggctgct ccacggaggt cagacctgag gggaccgtgtgctgctgtga ccacctgacc 120 tttttcgccc tgctcctgag acccaccttg gaccagtccacggtgcatat cctcacacgc 180 atctcccagg cgggctgtgg ggtctccatg atcttcctggccttcaccat tattctttat 240 gcctttctga ggctttcccg ggagaggttc aagtcagaagatgccccaaa gatccacgtg 300 gccctgggtg gcagcctgtt cctcctgaat ctggccttcttggtcaatgt ggggagtggc 360 tcaaaggggt ctgatgctgc ctgctgggcc cggggggctgtcttccacta cttcctgctc 420 tgtgccttca cctggatggg ccttgaagcc ttccacctctacctgctcgc tgtcagggtc 480 ttcaacacct acttcgggca ctacttcctg aagctgagcctggtgggctg gggcctgccc 540 gccctgatgg tcatcggcac tgggagtgcc aacagctacggcctctacac catccgtgat 600 agggagaacc gcacctctct ggagctatgc tggttccgtgaagggacaac catgtacgcc 660 ctctatatca ccgtccacgg ctacttcctc atcaccttcctctttggcat ggtggtcctg 720 gccctggtgg tctggaagat cttcaccctg tcccgtgctacagcggtcaa ggagcggggg 780 aagaaccggt gctcaccctg ctgggcctct cgagcctggtgggtgtga 828 36 275 PRT Homo sapiens 36 Met Thr Leu Thr Cys Val Phe TrpAsp Val Thr Lys Gly Thr Thr Gly 1 5 10 15 Asp Trp Ser Ser Glu Gly CysSer Thr Glu Val Arg Pro Glu Gly Thr 20 25 30 Val Cys Cys Cys Asp His LeuThr Phe Phe Ala Leu Leu Leu Arg Pro 35 40 45 Thr Leu Asp Gln Ser Thr ValHis Ile Leu Thr Arg Ile Ser Gln Ala 50 55 60 Gly Cys Gly Val Ser Met IlePhe Leu Ala Phe Thr Ile Ile Leu Tyr 65 70 75 80 Ala Phe Leu Arg Leu SerArg Glu Arg Phe Lys Ser Glu Asp Ala Pro 85 90 95 Lys Ile His Val Ala LeuGly Gly Ser Leu Phe Leu Leu Asn Leu Ala 100 105 110 Phe Leu Val Asn ValGly Ser Gly Ser Lys Gly Ser Asp Ala Ala Cys 115 120 125 Trp Ala Arg GlyAla Val Phe His Tyr Phe Leu Leu Cys Ala Phe Thr 130 135 140 Trp Met GlyLeu Glu Ala Phe His Leu Tyr Leu Leu Ala Val Arg Val 145 150 155 160 PheAsn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu Val Gly 165 170 175Trp Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser 180 185190 Tyr Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu 195200 205 Leu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr210 215 220 Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val ValLeu 225 230 235 240 Ala Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg AlaThr Ala Val 245 250 255 Lys Glu Arg Gly Lys Asn Arg Cys Ser Pro Cys TrpAla Ser Arg Ala 260 265 270 Trp Trp Val 275 37 1011 DNA Homo sapiens 37atggcccctt ctgcagcctg gcctccccga tctccccttt cacagggccc ccggctcggc 60ctgggagatg gcagcggcgt gttgaacaat cgcctggtgg gtttgagtgt gggacaaatg 120catgtcacca agctggctga gcctctggag atcgtcttct ctcaccagcg accgccccct 180aacatgaccc tcacctgtgt attctgggat gtgactaaag ggaccactgg agactggtct 240tctgagggct gctccacgga ggtcagacct gaggggaccg tgtgctgctg tgaccacctg 300acctttttcg ccctgctcct gagacccacc ttggaccagt ccacggtgca tatcctcaca 360cgcatctccc aggcgggctg tggggtctcc atgatcttcc tggccttcac cattattctt 420tatgcctttc tgaggctttc ccgggagagg ttcaagtcag aagatgcccc aaagatccac 480gtggccctgg gtggcagcct gttcctcctg aatctggcct tcttggtcaa tgtggggagt 540ggctcaaagg ggtctgatgc tgcctgctgg gcccgggggg ctgtcttcca ctacttcctg 600ctctgtgcct tcacctggat gggccttgaa gccttccacc tctacctgct cgctgtcagg 660gtcttcaaca cctacttcgg gcactacttc ctgaagctga gcctggtggg ctggggcctg 720cccgccctga tggtcatcgg cactgggagt gccaacagct acggcctcta caccatccgt 780gatagggaga accgcacctc tctggagcta tgctggttcc gtgaagggac aaccatgtac 840gccctctata tcaccgtcca cggctacttc ctcatcacct tcctctttgg catggtggtc 900ctggccctgg tggtctggaa gatcttcacc ctgtcccgtg ctacagcggt caaggagcgg 960gggaagaacc ggtgctcacc ctgctgggcc tctcgagcct ggtgggtgtg a 1011 38 336 PRTHomo sapiens 38 Met Ala Pro Ser Ala Ala Trp Pro Pro Arg Ser Pro Leu SerGln Gly 1 5 10 15 Pro Arg Leu Gly Leu Gly Asp Gly Ser Gly Val Leu AsnAsn Arg Leu 20 25 30 Val Gly Leu Ser Val Gly Gln Met His Val Thr Lys LeuAla Glu Pro 35 40 45 Leu Glu Ile Val Phe Ser His Gln Arg Pro Pro Pro AsnMet Thr Leu 50 55 60 Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr Thr GlyAsp Trp Ser 65 70 75 80 Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu GlyThr Val Cys Cys 85 90 95 Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu ArgPro Thr Leu Asp 100 105 110 Gln Ser Thr Val His Ile Leu Thr Arg Ile SerGln Ala Gly Cys Gly 115 120 125 Val Ser Met Ile Phe Leu Ala Phe Thr IleIle Leu Tyr Ala Phe Leu 130 135 140 Arg Leu Ser Arg Glu Arg Phe Lys SerGlu Asp Ala Pro Lys Ile His 145 150 155 160 Val Ala Leu Gly Gly Ser LeuPhe Leu Leu Asn Leu Ala Phe Leu Val 165 170 175 Asn Val Gly Ser Gly SerLys Gly Ser Asp Ala Ala Cys Trp Ala Arg 180 185 190 Gly Ala Val Phe HisTyr Phe Leu Leu Cys Ala Phe Thr Trp Met Gly 195 200 205 Leu Glu Ala PheHis Leu Tyr Leu Leu Ala Val Arg Val Phe Asn Thr 210 215 220 Tyr Phe GlyHis Tyr Phe Leu Lys Leu Ser Leu Val Gly Trp Gly Leu 225 230 235 240 ProAla Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser Tyr Gly Leu 245 250 255Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu Leu Cys Trp 260 265270 Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr Val His Gly 275280 285 Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val Val Leu Ala Leu Val290 295 300 Val Trp Lys Ile Phe Thr Leu Ser Arg Ala Thr Ala Val Lys GluArg 305 310 315 320 Gly Lys Asn Arg Cys Ser Pro Cys Trp Ala Ser Arg AlaTrp Trp Val 325 330 335 39 633 DNA Homo sapiens 39 atgggagctc cccatgggagctgtggcccc ttggggcctc ttatttctca ccccaggctt 60 tcccgggaga ggttcaagtcagaagatgcc ccaaagatcc acgtggccct gggtggcagc 120 ctgttcctcc tgaatctggccttcttggtc aatgtgggga gtggctcaaa ggggtctgat 180 gctgcctgct gggcccggggggctgtcttc cactacttcc tgctctgtgc cttcacctgg 240 atgggccttg aagccttccacctctacctg ctcgctgtca gggtcttcaa cacctacttc 300 gggcactact tcctgaagctgagcctggtg ggctggggcc tgcccgccct gatggtcatc 360 ggcactggga gtgccaacagctacggcctc tacaccatcc gtgataggga gaaccgcacc 420 tctctggagc tatgctggttccgtgaaggg acaaccatgt acgccctcta tatcaccgtc 480 cacggctact tcctcatcaccttcctcttt ggcatggtgg tcctggccct ggtggtctgg 540 aagatcttca ccctgtcccgtgctacagcg gtcaaggagc gggggaagaa ccggtgctca 600 ccctgctggg cctctcgagcctggtgggtg tga 633 40 210 PRT Homo sapiens 40 Met Gly Ala Pro His GlySer Cys Gly Pro Leu Gly Pro Leu Ile Ser 1 5 10 15 His Pro Arg Leu SerArg Glu Arg Phe Lys Ser Glu Asp Ala Pro Lys 20 25 30 Ile His Val Ala LeuGly Gly Ser Leu Phe Leu Leu Asn Leu Ala Phe 35 40 45 Leu Val Asn Val GlySer Gly Ser Lys Gly Ser Asp Ala Ala Cys Trp 50 55 60 Ala Arg Gly Ala ValPhe His Tyr Phe Leu Leu Cys Ala Phe Thr Trp 65 70 75 80 Met Gly Leu GluAla Phe His Leu Tyr Leu Leu Ala Val Arg Val Phe 85 90 95 Asn Thr Tyr PheGly His Tyr Phe Leu Lys Leu Ser Leu Val Gly Trp 100 105 110 Gly Leu ProAla Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser Tyr 115 120 125 Gly LeuTyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu Leu 130 135 140 CysTrp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr Val 145 150 155160 His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val Val Leu Ala 165170 175 Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg Ala Thr Ala Val Lys180 185 190 Glu Arg Gly Lys Asn Arg Cys Ser Pro Cys Trp Ala Ser Arg AlaTrp 195 200 205 Trp Val 210 41 420 DNA Homo sapiens 41 atggggcaaatgaaacatgt ctttgaggtc actttggcat taaagagaca ccagactgga 60 gccaggtggcggcccctccc acagcgggag agccagggat tgatgggtgg aaatgggaga 120 ggcaccttcacagacagaaa agctcagcca ggggacttcc tgggtttgct ggccagaggt 180 accactcccagtcccaccac agctgccccc tcctccagat gctggttccg tgaagggaca 240 accatgtacgccctctatat caccgtccac ggctacttcc tcatcacctt cctctttggc 300 atggtggtcctggccctggt ggtctggaag atcttcaccc tgtcccgtgc tacagcggtc 360 aaggagcgggggaagaaccg gtgctcaccc tgctgggcct ctcgagcctg gtgggtgtga 420 42 139 PRTHomo sapiens 42 Met Gly Gln Met Lys His Val Phe Glu Val Thr Leu Ala LeuLys Arg 1 5 10 15 His Gln Thr Gly Ala Arg Trp Arg Pro Leu Pro Gln ArgGlu Ser Gln 20 25 30 Gly Leu Met Gly Gly Asn Gly Arg Gly Thr Phe Thr AspArg Lys Ala 35 40 45 Gln Pro Gly Asp Phe Leu Gly Leu Leu Ala Arg Gly ThrThr Pro Ser 50 55 60 Pro Thr Thr Ala Ala Pro Ser Ser Arg Cys Trp Phe ArgGlu Gly Thr 65 70 75 80 Thr Met Tyr Ala Leu Tyr Ile Thr Val His Gly TyrPhe Leu Ile Thr 85 90 95 Phe Leu Phe Gly Met Val Val Leu Ala Leu Val ValTrp Lys Ile Phe 100 105 110 Thr Leu Ser Arg Ala Thr Ala Val Lys Glu ArgGly Lys Asn Arg Cys 115 120 125 Ser Pro Cys Trp Ala Ser Arg Ala Trp TrpVal 130 135 43 1650 DNA Homo sapiens 43 atggcgacgc ccaggggcct gggggccctgctcctgctcc tcctgctccc gacctcaggt 60 caggaaaagc ccaccgaagg gccaagaaacacctgcctgg ggagcaacaa catgtacgac 120 atcttcaact tgaatgacaa ggctttgtgcttcaccaagt gcaggcagtc gggcagcgac 180 tcctgcaatg tggaaaactt gcagagatactggctaaact acgaggccca tctgatgaag 240 gaaggtttga cgcagaaggt gaacacgcctttcctgaagg ctttggtcca gaacctcagc 300 accaacactg cagaagactt ctatttctctctggagccct ctcaggttcc gaggcaggtg 360 atgaaggacg aggacaagcc ccctgacagagtgcgacttc ccaagagcct ttttcgatcc 420 ctgccaggca acaggtctgt ggtccgcttggccgtcacca ttctggacat tggtccaggg 480 actctcttca agggcccccg gctcggcctgggagatggca gcggcgtgtt gaacaatcgc 540 ctggtgggtt tgagtgtggg acaaatgcatgtcaccaagc tggctgagcc tctggagatc 600 gtcttctctc accagcgacc gccccctaacatgaccctca cctgtgtatt ctgggatgtg 660 actaaaggga ccactggaga ctggtcttctgagggctgct ccacggaggt cagacctgag 720 gggaccgtgt gctgctgtga ccacctgacctttttcgccc tgctcctgag acccaccttg 780 gaccagtcca cggtgcatat cctcacacgcatctcccagg cgggctgtgg ggtctccatg 840 atcttcctgg ccttcaccat tattctttatgcctttctga ggctttcccg ggagaggttc 900 aagtcagaag atgccccaaa gatccacgtggccctgggtg gcagcctgtt cctcctgaat 960 ctggccttct tggtcaatgt ggggagtggctcaaaggggt ctgatgctgc ctgctgggcc 1020 cggggggctg tcttccacta cttcctgctctgtgccttca cctggatggg ccttgaagcc 1080 ttccacctct acctgctcgc tgtcagggtcttcaacacct acttcgggca ctacttcctg 1140 aagctgagcc tggtgggctg gggcctgcccgccctgatgg tcatcggcac tgggagtgcc 1200 aacagctacg gcctctacac catccgtgatagggagaacc gcacctctct ggagctatgc 1260 tggttccgtg aagggacaac catgtacgccctctatatca ccgtccacgg ctacttcctc 1320 atcaccttcc tctttggcat ggtggtcctggccctggtgg tctggaagat cttcaccctg 1380 tcccgtgcta cagcggtcaa ggagcgggggaagaaccgga agaaggtgct caccctgctg 1440 ggcctctcga gcctggtggg tgtgacatgggggttggcca tcttcacccc gttgggcctc 1500 tccaccgtct acatctttgc acttttcaactccttgcaag gtgtcttcat ctgctgctgg 1560 ttcaccatcc tttacctccc aagtcagagcaccacagtct cctcctctac tgcaagattg 1620 gaccaggccc actccgcatc tcaagaatag1650 44 549 PRT Homo sapiens 44 Met Ala Thr Pro Arg Gly Leu Gly Ala LeuLeu Leu Leu Leu Leu Leu 1 5 10 15 Pro Thr Ser Gly Gln Glu Lys Pro ThrGlu Gly Pro Arg Asn Thr Cys 20 25 30 Leu Gly Ser Asn Asn Met Tyr Asp IlePhe Asn Leu Asn Asp Lys Ala 35 40 45 Leu Cys Phe Thr Lys Cys Arg Gln SerGly Ser Asp Ser Cys Asn Val 50 55 60 Glu Asn Leu Gln Arg Tyr Trp Leu AsnTyr Glu Ala His Leu Met Lys 65 70 75 80 Glu Gly Leu Thr Gln Lys Val AsnThr Pro Phe Leu Lys Ala Leu Val 85 90 95 Gln Asn Leu Ser Thr Asn Thr AlaGlu Asp Phe Tyr Phe Ser Leu Glu 100 105 110 Pro Ser Gln Val Pro Arg GlnVal Met Lys Asp Glu Asp Lys Pro Pro 115 120 125 Asp Arg Val Arg Leu ProLys Ser Leu Phe Arg Ser Leu Pro Gly Asn 130 135 140 Arg Ser Val Val ArgLeu Ala Val Thr Ile Leu Asp Ile Gly Pro Gly 145 150 155 160 Thr Leu PheLys Gly Pro Arg Leu Gly Leu Gly Asp Gly Ser Gly Val 165 170 175 Leu AsnAsn Arg Leu Val Gly Leu Ser Val Gly Gln Met His Val Thr 180 185 190 LysLeu Ala Glu Pro Leu Glu Ile Val Phe Ser His Gln Arg Pro Pro 195 200 205Pro Asn Met Thr Leu Thr Cys Val Phe Trp Asp Val Thr Lys Gly Thr 210 215220 Thr Gly Asp Trp Ser Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu 225230 235 240 Gly Thr Val Cys Cys Cys Asp His Leu Thr Phe Phe Ala Leu LeuLeu 245 250 255 Arg Pro Thr Leu Asp Gln Ser Thr Val His Ile Leu Thr ArgIle Ser 260 265 270 Gln Ala Gly Cys Gly Val Ser Met Ile Phe Leu Ala PheThr Ile Ile 275 280 285 Leu Tyr Ala Phe Leu Arg Leu Ser Arg Glu Arg PheLys Ser Glu Asp 290 295 300 Ala Pro Lys Ile His Val Ala Leu Gly Gly SerLeu Phe Leu Leu Asn 305 310 315 320 Leu Ala Phe Leu Val Asn Val Gly SerGly Ser Lys Gly Ser Asp Ala 325 330 335 Ala Cys Trp Ala Arg Gly Ala ValPhe His Tyr Phe Leu Leu Cys Ala 340 345 350 Phe Thr Trp Met Gly Leu GluAla Phe His Leu Tyr Leu Leu Ala Val 355 360 365 Arg Val Phe Asn Thr TyrPhe Gly His Tyr Phe Leu Lys Leu Ser Leu 370 375 380 Val Gly Trp Gly LeuPro Ala Leu Met Val Ile Gly Thr Gly Ser Ala 385 390 395 400 Asn Ser TyrGly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser 405 410 415 Leu GluLeu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr 420 425 430 IleThr Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val 435 440 445Val Leu Ala Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg Ala Thr 450 455460 Ala Val Lys Glu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr Leu Leu 465470 475 480 Gly Leu Ser Ser Leu Val Gly Val Thr Trp Gly Leu Ala Ile PheThr 485 490 495 Pro Leu Gly Leu Ser Thr Val Tyr Ile Phe Ala Leu Phe AsnSer Leu 500 505 510 Gln Gly Val Phe Ile Cys Cys Trp Phe Thr Ile Leu TyrLeu Pro Ser 515 520 525 Gln Ser Thr Thr Val Ser Ser Ser Thr Ala Arg LeuAsp Gln Ala His 530 535 540 Ser Ala Ser Gln Glu 545 45 1020 DNA Homosapiens 45 atgaccctca cctgtgtatt ctgggatgtg actaaaggga ccactggagactggtcttct 60 gagggctgct ccacggaggt cagacctgag gggaccgtgt gctgctgtgaccacctgacc 120 tttttcgccc tgctcctgag acccaccttg gaccagtcca cggtgcatatcctcacacgc 180 atctcccagg cgggctgtgg ggtctccatg atcttcctgg ccttcaccattattctttat 240 gcctttctga ggctttcccg ggagaggttc aagtcagaag atgccccaaagatccacgtg 300 gccctgggtg gcagcctgtt cctcctgaat ctggccttct tggtcaatgtggggagtggc 360 tcaaaggggt ctgatgctgc ctgctgggcc cggggggctg tcttccactacttcctgctc 420 tgtgccttca cctggatggg ccttgaagcc ttccacctct acctgctcgctgtcagggtc 480 ttcaacacct acttcgggca ctacttcctg aagctgagcc tggtgggctggggcctgccc 540 gccctgatgg tcatcggcac tgggagtgcc aacagctacg gcctctacaccatccgtgat 600 agggagaacc gcacctctct ggagctatgc tggttccgtg aagggacaaccatgtacgcc 660 ctctatatca ccgtccacgg ctacttcctc atcaccttcc tctttggcatggtggtcctg 720 gccctggtgg tctggaagat cttcaccctg tcccgtgcta cagcggtcaaggagcggggg 780 aagaaccgga agaaggtgct caccctgctg ggcctctcga gcctggtgggtgtgacatgg 840 gggttggcca tcttcacccc gttgggcctc tccaccgtct acatctttgcacttttcaac 900 tccttgcaag gtgtcttcat ctgctgctgg ttcaccatcc tttacctcccaagtcagagc 960 accacagtct cctcctctac tgcaagattg gaccaggccc actccgcatctcaagaatag 1020 46 339 PRT Homo sapiens 46 Met Thr Leu Thr Cys Val PheTrp Asp Val Thr Lys Gly Thr Thr Gly 1 5 10 15 Asp Trp Ser Ser Glu GlyCys Ser Thr Glu Val Arg Pro Glu Gly Thr 20 25 30 Val Cys Cys Cys Asp HisLeu Thr Phe Phe Ala Leu Leu Leu Arg Pro 35 40 45 Thr Leu Asp Gln Ser ThrVal His Ile Leu Thr Arg Ile Ser Gln Ala 50 55 60 Gly Cys Gly Val Ser MetIle Phe Leu Ala Phe Thr Ile Ile Leu Tyr 65 70 75 80 Ala Phe Leu Arg LeuSer Arg Glu Arg Phe Lys Ser Glu Asp Ala Pro 85 90 95 Lys Ile His Val AlaLeu Gly Gly Ser Leu Phe Leu Leu Asn Leu Ala 100 105 110 Phe Leu Val AsnVal Gly Ser Gly Ser Lys Gly Ser Asp Ala Ala Cys 115 120 125 Trp Ala ArgGly Ala Val Phe His Tyr Phe Leu Leu Cys Ala Phe Thr 130 135 140 Trp MetGly Leu Glu Ala Phe His Leu Tyr Leu Leu Ala Val Arg Val 145 150 155 160Phe Asn Thr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu Val Gly 165 170175 Trp Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser 180185 190 Tyr Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu195 200 205 Leu Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr IleThr 210 215 220 Val His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met ValVal Leu 225 230 235 240 Ala Leu Val Val Trp Lys Ile Phe Thr Leu Ser ArgAla Thr Ala Val 245 250 255 Lys Glu Arg Gly Lys Asn Arg Lys Lys Val LeuThr Leu Leu Gly Leu 260 265 270 Ser Ser Leu Val Gly Val Thr Trp Gly LeuAla Ile Phe Thr Pro Leu 275 280 285 Gly Leu Ser Thr Val Tyr Ile Phe AlaLeu Phe Asn Ser Leu Gln Gly 290 295 300 Val Phe Ile Cys Cys Trp Phe ThrIle Leu Tyr Leu Pro Ser Gln Ser 305 310 315 320 Thr Thr Val Ser Ser SerThr Ala Arg Leu Asp Gln Ala His Ser Ala 325 330 335 Ser Gln Glu 47 1203DNA Homo sapiens 47 atggcccctt ctgcagcctg gcctccccga tctcccctttcacagggccc ccggctcggc 60 ctgggagatg gcagcggcgt gttgaacaat cgcctggtgggtttgagtgt gggacaaatg 120 catgtcacca agctggctga gcctctggag atcgtcttctctcaccagcg accgccccct 180 aacatgaccc tcacctgtgt attctgggat gtgactaaagggaccactgg agactggtct 240 tctgagggct gctccacgga ggtcagacct gaggggaccgtgtgctgctg tgaccacctg 300 acctttttcg ccctgctcct gagacccacc ttggaccagtccacggtgca tatcctcaca 360 cgcatctccc aggcgggctg tggggtctcc atgatcttcctggccttcac cattattctt 420 tatgcctttc tgaggctttc ccgggagagg ttcaagtcagaagatgcccc aaagatccac 480 gtggccctgg gtggcagcct gttcctcctg aatctggccttcttggtcaa tgtggggagt 540 ggctcaaagg ggtctgatgc tgcctgctgg gcccggggggctgtcttcca ctacttcctg 600 ctctgtgcct tcacctggat gggccttgaa gccttccacctctacctgct cgctgtcagg 660 gtcttcaaca cctacttcgg gcactacttc ctgaagctgagcctggtggg ctggggcctg 720 cccgccctga tggtcatcgg cactgggagt gccaacagctacggcctcta caccatccgt 780 gatagggaga accgcacctc tctggagcta tgctggttccgtgaagggac aaccatgtac 840 gccctctata tcaccgtcca cggctacttc ctcatcaccttcctctttgg catggtggtc 900 ctggccctgg tggtctggaa gatcttcacc ctgtcccgtgctacagcggt caaggagcgg 960 gggaagaacc ggaagaaggt gctcaccctg ctgggcctctcgagcctggt gggtgtgaca 1020 tgggggttgg ccatcttcac cccgttgggc ctctccaccgtctacatctt tgcacttttc 1080 aactccttgc aaggtgtctt catctgctgc tggttcaccatcctttacct cccaagtcag 1140 agcaccacag tctcctcctc tactgcaaga ttggaccaggcccactccgc atctcaagaa 1200 tag 1203 48 400 PRT Homo sapiens 48 Met AlaPro Ser Ala Ala Trp Pro Pro Arg Ser Pro Leu Ser Gln Gly 1 5 10 15 ProArg Leu Gly Leu Gly Asp Gly Ser Gly Val Leu Asn Asn Arg Leu 20 25 30 ValGly Leu Ser Val Gly Gln Met His Val Thr Lys Leu Ala Glu Pro 35 40 45 LeuGlu Ile Val Phe Ser His Gln Arg Pro Pro Pro Asn Met Thr Leu 50 55 60 ThrCys Val Phe Trp Asp Val Thr Lys Gly Thr Thr Gly Asp Trp Ser 65 70 75 80Ser Glu Gly Cys Ser Thr Glu Val Arg Pro Glu Gly Thr Val Cys Cys 85 90 95Cys Asp His Leu Thr Phe Phe Ala Leu Leu Leu Arg Pro Thr Leu Asp 100 105110 Gln Ser Thr Val His Ile Leu Thr Arg Ile Ser Gln Ala Gly Cys Gly 115120 125 Val Ser Met Ile Phe Leu Ala Phe Thr Ile Ile Leu Tyr Ala Phe Leu130 135 140 Arg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp Ala Pro Lys IleHis 145 150 155 160 Val Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn Leu AlaPhe Leu Val 165 170 175 Asn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala AlaCys Trp Ala Arg 180 185 190 Gly Ala Val Phe His Tyr Phe Leu Leu Cys AlaPhe Thr Trp Met Gly 195 200 205 Leu Glu Ala Phe His Leu Tyr Leu Leu AlaVal Arg Val Phe Asn Thr 210 215 220 Tyr Phe Gly His Tyr Phe Leu Lys LeuSer Leu Val Gly Trp Gly Leu 225 230 235 240 Pro Ala Leu Met Val Ile GlyThr Gly Ser Ala Asn Ser Tyr Gly Leu 245 250 255 Tyr Thr Ile Arg Asp ArgGlu Asn Arg Thr Ser Leu Glu Leu Cys Trp 260 265 270 Phe Arg Glu Gly ThrThr Met Tyr Ala Leu Tyr Ile Thr Val His Gly 275 280 285 Tyr Phe Leu IleThr Phe Leu Phe Gly Met Val Val Leu Ala Leu Val 290 295 300 Val Trp LysIle Phe Thr Leu Ser Arg Ala Thr Ala Val Lys Glu Arg 305 310 315 320 GlyLys Asn Arg Lys Lys Val Leu Thr Leu Leu Gly Leu Ser Ser Leu 325 330 335Val Gly Val Thr Trp Gly Leu Ala Ile Phe Thr Pro Leu Gly Leu Ser 340 345350 Thr Val Tyr Ile Phe Ala Leu Phe Asn Ser Leu Gln Gly Val Phe Ile 355360 365 Cys Cys Trp Phe Thr Ile Leu Tyr Leu Pro Ser Gln Ser Thr Thr Val370 375 380 Ser Ser Ser Thr Ala Arg Leu Asp Gln Ala His Ser Ala Ser GlnGlu 385 390 395 400 49 825 DNA Homo sapiens 49 atgggagctc cccatgggagctgtggcccc ttggggcctc ttatttctca ccccaggctt 60 tcccgggaga ggttcaagtcagaagatgcc ccaaagatcc acgtggccct gggtggcagc 120 ctgttcctcc tgaatctggccttcttggtc aatgtgggga gtggctcaaa ggggtctgat 180 gctgcctgct gggcccggggggctgtcttc cactacttcc tgctctgtgc cttcacctgg 240 atgggccttg aagccttccacctctacctg ctcgctgtca gggtcttcaa cacctacttc 300 gggcactact tcctgaagctgagcctggtg ggctggggcc tgcccgccct gatggtcatc 360 ggcactggga gtgccaacagctacggcctc tacaccatcc gtgataggga gaaccgcacc 420 tctctggagc tatgctggttccgtgaaggg acaaccatgt acgccctcta tatcaccgtc 480 cacggctact tcctcatcaccttcctcttt ggcatggtgg tcctggccct ggtggtctgg 540 aagatcttca ccctgtcccgtgctacagcg gtcaaggagc gggggaagaa ccggaagaag 600 gtgctcaccc tgctgggcctctcgagcctg gtgggtgtga catgggggtt ggccatcttc 660 accccgttgg gcctctccaccgtctacatc tttgcacttt tcaactcctt gcaaggtgtc 720 ttcatctgct gctggttcaccatcctttac ctcccaagtc agagcaccac agtctcctcc 780 tctactgcaa gattggaccaggcccactcc gcatctcaag aatag 825 50 274 PRT Homo sapiens 50 Met Gly AlaPro His Gly Ser Cys Gly Pro Leu Gly Pro Leu Ile Ser 1 5 10 15 His ProArg Leu Ser Arg Glu Arg Phe Lys Ser Glu Asp Ala Pro Lys 20 25 30 Ile HisVal Ala Leu Gly Gly Ser Leu Phe Leu Leu Asn Leu Ala Phe 35 40 45 Leu ValAsn Val Gly Ser Gly Ser Lys Gly Ser Asp Ala Ala Cys Trp 50 55 60 Ala ArgGly Ala Val Phe His Tyr Phe Leu Leu Cys Ala Phe Thr Trp 65 70 75 80 MetGly Leu Glu Ala Phe His Leu Tyr Leu Leu Ala Val Arg Val Phe 85 90 95 AsnThr Tyr Phe Gly His Tyr Phe Leu Lys Leu Ser Leu Val Gly Trp 100 105 110Gly Leu Pro Ala Leu Met Val Ile Gly Thr Gly Ser Ala Asn Ser Tyr 115 120125 Gly Leu Tyr Thr Ile Arg Asp Arg Glu Asn Arg Thr Ser Leu Glu Leu 130135 140 Cys Trp Phe Arg Glu Gly Thr Thr Met Tyr Ala Leu Tyr Ile Thr Val145 150 155 160 His Gly Tyr Phe Leu Ile Thr Phe Leu Phe Gly Met Val ValLeu Ala 165 170 175 Leu Val Val Trp Lys Ile Phe Thr Leu Ser Arg Ala ThrAla Val Lys 180 185 190 Glu Arg Gly Lys Asn Arg Lys Lys Val Leu Thr LeuLeu Gly Leu Ser 195 200 205 Ser Leu Val Gly Val Thr Trp Gly Leu Ala IlePhe Thr Pro Leu Gly 210 215 220 Leu Ser Thr Val Tyr Ile Phe Ala Leu PheAsn Ser Leu Gln Gly Val 225 230 235 240 Phe Ile Cys Cys Trp Phe Thr IleLeu Tyr Leu Pro Ser Gln Ser Thr 245 250 255 Thr Val Ser Ser Ser Thr AlaArg Leu Asp Gln Ala His Ser Ala Ser 260 265 270 Gln Glu 51 612 DNA Homosapiens 51 atggggcaaa tgaaacatgt ctttgaggtc actttggcat taaagagacaccagactgga 60 gccaggtggc ggcccctccc acagcgggag agccagggat tgatgggtggaaatgggaga 120 ggcaccttca cagacagaaa agctcagcca ggggacttcc tgggtttgctggccagaggt 180 accactccca gtcccaccac agctgccccc tcctccagat gctggttccgtgaagggaca 240 accatgtacg ccctctatat caccgtccac ggctacttcc tcatcaccttcctctttggc 300 atggtggtcc tggccctggt ggtctggaag atcttcaccc tgtcccgtgctacagcggtc 360 aaggagcggg ggaagaaccg gaagaaggtg ctcaccctgc tgggcctctcgagcctggtg 420 ggtgtgacat gggggttggc catcttcacc ccgttgggcc tctccaccgtctacatcttt 480 gcacttttca actccttgca aggtgtcttc atctgctgct ggttcaccatcctttacctc 540 ccaagtcaga gcaccacagt ctcctcctct actgcaagat tggaccaggcccactccgca 600 tctcaagaat ag 612 52 203 PRT Homo sapiens 52 Met Gly GlnMet Lys His Val Phe Glu Val Thr Leu Ala Leu Lys Arg 1 5 10 15 His GlnThr Gly Ala Arg Trp Arg Pro Leu Pro Gln Arg Glu Ser Gln 20 25 30 Gly LeuMet Gly Gly Asn Gly Arg Gly Thr Phe Thr Asp Arg Lys Ala 35 40 45 Gln ProGly Asp Phe Leu Gly Leu Leu Ala Arg Gly Thr Thr Pro Ser 50 55 60 Pro ThrThr Ala Ala Pro Ser Ser Arg Cys Trp Phe Arg Glu Gly Thr 65 70 75 80 ThrMet Tyr Ala Leu Tyr Ile Thr Val His Gly Tyr Phe Leu Ile Thr 85 90 95 PheLeu Phe Gly Met Val Val Leu Ala Leu Val Val Trp Lys Ile Phe 100 105 110Thr Leu Ser Arg Ala Thr Ala Val Lys Glu Arg Gly Lys Asn Arg Lys 115 120125 Lys Val Leu Thr Leu Leu Gly Leu Ser Ser Leu Val Gly Val Thr Trp 130135 140 Gly Leu Ala Ile Phe Thr Pro Leu Gly Leu Ser Thr Val Tyr Ile Phe145 150 155 160 Ala Leu Phe Asn Ser Leu Gln Gly Val Phe Ile Cys Cys TrpPhe Thr 165 170 175 Ile Leu Tyr Leu Pro Ser Gln Ser Thr Thr Val Ser SerSer Thr Ala 180 185 190 Arg Leu Asp Gln Ala His Ser Ala Ser Gln Glu 195200 53 4036 DNA Homo sapiens 53 ggccagaggg ccagacagcc acagagctcctggcgtgggc aaggctggcc aaggatggcg 60 acgcccaggg gcctgggggc cctgctcctgctcctcctgc tcccgacctc aggtcaggaa 120 aagcccaccg aagggccaag aaacacctgcctggggagca acaacatgta cgacatcttc 180 aacttgaatg acaaggcttt gtgcttcaccaagtgcaggc agtcgggcag cgactcctgc 240 aatgtggaaa acttgcagag atactggctaaactacgagg cccatctgat gaaggaaggt 300 ttgacgcaga aggtgaacac gcctttcctgaaggctttgg tccagaacct cagcaccaac 360 actgcagaag acttctattt ctctctggagccctctcagg ttccgaggca ggtgatgaag 420 gacgaggaca agccccctga cagagtgcgacttcccaaga gcctttttcg atccctgcca 480 ggcaacaggt ctgtggtccg cttggccgtcaccattctgg acattggtcc agggactctc 540 ttcacacatg tgtataccag gtatgtgcacccagaggtgt gcatccactc ctgtgcagac 600 gtgtgtaccc ctgagggcta gtgtgctccccccaccagcc tcctttctac cgaatgcaca 660 ctcacgctaa gaccctcagg ggcacgctatcctccccgct gacttccatt tcttggctga 720 tcttggcccc atgccccctc tagttaagagggcagaggag ctctggaggc cagcaatgga 780 gagctgtcag gtgcacagct ttgcagccagttgacctggc ccagcccaag caggagacca 840 ctgggagcag cagggaggag gctgcctgtgactccttggc tccctggtcc cctggtctcg 900 aactctgccc tccaagcaaa ggccatgggttcctggaggc tcctaggaac cccagcgttg 960 gtgggttggg atggcccctt ctgcagcctggcctccccga tctccccttt cacagggccc 1020 ccggctcggc ctgggagatg gcagcggcgtgttgaacaat cgcctggtgg gtttgagtgt 1080 gggacaaatg catgtcacca agctggctgagcctctggag atcgtcttct ctcaccagcg 1140 accgccccct gtgagtcccc tgctcaggcctggcagccac tgcagggcag acagaacatg 1200 accctcacct gtgtattctg ggatgtgactaaagggacca ctggagactg gtcttctgag 1260 ggctgctcca cggaggtcag acctgaggggaccgtgtgct gctgtgacca cctgaccttt 1320 ttcgccctgc tcctgagacc caccttggaccagtccacgg tgcatatcct cacacgcatc 1380 tcccaggcgg gctgtggggt ctccatgatcttcctggcct tcaccattat tctttatgcc 1440 tttctgcatt ccaggtgttt ttttcttctcttcccaaggc tgcctaatct ctagccagtg 1500 tctggctttt gactgatagg tgtgttgctcagttactttg ggcccgtgta cgtttgtgtg 1560 tcacctccat cccataattt taagtacatgcatgatatgc agcccatatg catgaacctt 1620 aagtagctaa ttatcataca gggttatgtgaaagaaactt tttctctcta atgtaaatgc 1680 ccatctctga agagctgccc cttactggtttggtccggat cttgccggcc acggggtccc 1740 ttttttatgt cacttttgtc ttgcctgctgaacctctgct tttcatctca cttcttgctc 1800 acccgtccca ttcaccgtgc ttctattctctgcttttact tattctgccc tttatccaac 1860 ttttaattcc ctttgctatt ctcctgcctcattttctggc ctcattttcc ctattatcct 1920 gcctcacatt gatcaaggga tgaggctggcaggatccgga acccacaggg ccccgtgggc 1980 catgagaggc tcctggactt gaacctcaggacactcccac tctggctgcc ggcagggatg 2040 gaagctggat gagcaggcag gagctggcagtgggggtgga gagccatagg ctattggggt 2100 ggacaggctt gggtgcctca tgggagctccccatgggagc tgtggcccct tggggcctct 2160 tatttctcac cccaggcttt cccgggagaggttcaagtca gaagatgccc caaagatcca 2220 cgtggccctg ggtggcagcc tgttcctcctgaatctggcc ttcttggtca atgtggggag 2280 tggctcaaag gggtctgatg ctgcctgctgggcccggggg gctgtcttcc actacttcct 2340 gctctgtgcc ttcacctgga tgggccttgaagccttccac ctctacctgc tcgctgtcag 2400 ggtcttcaac acctacttcg ggcactacttcctgaagctg agcctggtgg gctggggcct 2460 gcccgccctg atggtcatcg gcactgggagtgccaacagc tacggcctct acaccatccg 2520 tgatagggag aaccgcacct ctctggagctgtggggactg cagcggactg gcagtcacaa 2580 gcccatctaa ttagcggtca gttactatccttcaggaggg catccacaga gctgccaggt 2640 gtatgatttt ataggagaag cagaaatctaggtgtttata ccaaagcttc tgattttaaa 2700 ggcggccact aattccgttt ttttcwcaatgtaatatggg gcaaatgaaa catgtctttg 2760 aggtcacttt ggcattaaag agacaccagactggagccag gtggcggccc ctcccacagc 2820 gggagagcca gggattgatg ggtggaaatgggagaggcac cttcacagac agaaaagctc 2880 agccagggga cttcctgggt ttgctggccagaggtaccac tcccagtccc accacagctg 2940 ccccctcctc cagatgctgg ttccgtgaagggacaaccat gtacgccctc tatatcaccg 3000 tccacggcta cttcctcatc accttcctctttggcatggt ggtcctggcc ctggtggtct 3060 ggaagatctt caccctgtcc cgtgctacagcggtcaagga gcgggggaag aaccggaaga 3120 aggtgctcac cctgctgggc ctctcgagcctggtgggtgt gacatggggg ttggccatct 3180 tcaccccgtt gggcctctcc accgtctacatctttgcact tttcaactcc ttgcaaggtg 3240 aggcccctgc accagggagg tgatgggctgtgttgtctgt cccaggaggt attgggaggt 3300 ggggaagagg gtggtttgca agacacaggactctgttcag gctagctgaa gtcaaggatg 3360 ttgatttcaa atactcagag caaggatccagggcagcaaa gtttggctgc tgtattagtc 3420 cgtttgtgtt acttgcaagt tgggtgtccatcgtccatct ctggtccaat cagctgcgac 3480 cagaagggca gaatcatgtg atatgatgtccacatgacat ggatgggatc tccagggatt 3540 cttcatctgc tgctggttca ccatcctttacctcccaagt cagagcacca cagtctcctc 3600 ctctactgca agattggacc aggcccactccgcatctcaa gaataggaag gcacggccct 3660 gcaatatgga ctcagctctg gctctctgtgtgaccttggg cagctccgtg cctctctctg 3720 tactccctca gtttccttct ctgtacaatgtggctgggga gggagaggat gggaccaggt 3780 tggaccacgt ggcatcagag gtcccatccagatccaacta taggtccaag agtccacgta 3840 agcaggtttg caaggctcta aagttcctatagtcctgaga ccccctgcca gcaaagagtg 3900 acagtcacct ccatgccctg ccctcattgcaaagccctca ctcaccttct ggtctcagca 3960 agggaggaga gtctgttgct ggcatagccctggaaggagc ccccagcctc tccccttctc 4020 cttcttgtca ctggcc 4036

What is claimed is:
 1. An isolated nucleic acid molecule comprising atleast 22 contiguous bases of nucleotide sequence first disclosed in theNGPCR polynucleotide described in SEQ ID NO:
 43. 2. An isolated nucleicacid molecule comprising a nucleotide sequence that: (a) encodes theamino acid sequence shown in SEQ ID NO: 44; and (b) hybridizes understringent conditions to the nucleotide sequence of SEQ ID NO: 43 or thecomplement thereof.
 3. An isolated nucleic acid molecule comprising anucleotide sequence that encodes the amino acid sequence shown in SEQ IDNO:
 44. 4. An isolated nucleic acid molecule comprising a nucleotidesequence that encodes the amino acid sequence shown in SEQ ID NO:4. 5.An isolated nucleic acid molecule comprising a nucleotide sequence thatencodes the amino acid sequence shown in SEQ ID NO:34.